Non-racemic hexafluoreleucine, and methods of making and using it

ABSTRACT

One aspect of the invention relates to hexafluoroleucine and congeners thereof, and methods of making the compounds. Another aspect of the nvention relates to the synthesis of protein cores comprising hexafluoroleucine and congeners thereof. Certain peptides comprising hexafluorleucine and congeners thereof have been characterized using comparative biophysical studies. In general, the fluorinated peptides show higher thermal stability and enhanced resistance to chemical denaturation. Further, mixed hydrocarbonfluorocarbon cores self-sort into homogeneous bundles, suggesting new avenues for the design and manipulation of protein-protein interfaces.

BACKGROUND OF THE INVENTION

[0001] Proteins fold to adopt unique three dimensional structures,usually as a result of multiple non-covalent interactions thatcontribute to their conformational stability. Creighton, T. E. Proteins:Structures and Molecular Properties; 2nd ed.; W. H. Freeman: New York,1993. Removal of hydrophobic surface area from aqueous solvent plays adominant role in stabilizing protein structures. Tanford, C. Science1978, 200, 1012-1018; and Kauzmann, W. Adv. Protein Chem. 1959, 14,1-63. For instance, a buried leucine or phenylalanine residue cancontribute ˜2-5 kcal/mol in stability when compared to alanine. Althoughhydrogen bonds and salt bridges, when present in hydrophobicenvironments, can contribute as much as 3 kcal/mol to protein stability,solvent exposed electrostatic interactions contribute far less, usually0.5 kcal/mol. Yu, Y. H.; Monera, O. D.; Hodges, R. S.; Privalov, P. L.J. Mol. Biol. 1996, 255, 367-372; and Lumb, K. J.; Kim, P. S. Science1995, 268, 436-439. Hydrogen bonds between small polar side chains andbackbone amides can be worth 1-2 kcal/mol, as seen in the case ofN-terminal helical caps. Aurora, R.; Rose, G. D. Protein Sci. 1998, 7,21-38. The energetic balance of these intramolecular forces andinteractions with the solvent determines the shape and the stability ofthe fold.

[0002] While electrostatic interactions in designed structures canprovide conformational specificity at the expense of thermodynamicstability, hydrophobic interactions afford a very powerful driving forcefor stabilizing structures. Recent studies have focused on theintroduction of non-proteinogenic, fluorine containing amino acids as ameans for increasing hydrophobicity, without significant concurrentalteration of protein structure. Bilgicer, B.; Fichera, A.; Kumar, K. J.Am. Chem. Soc. 2001, 123, 4393-4399; and Tang, Y.; Ghirlanda, G.;Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, W. A.; DeGrado, W. F.;Tirrell, D. A. Biochemistry 2001, 40, 2790-2796. The estimated averagevolumes of CH₂ and CH₃ groups are 27 and 54 Å³, respectively, ascompared to the much larger 38 and 92 Å³ for CF₂ and CF₃ groups.Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Biochim. BiophysicaActa 1977, 470, 185-201. Given that the hydrophobic effect is roughlyproportional to the solvent exposed surface area, the large size andvolume of trifluoromethyl groups, in combination with the lowpolarizability of fluorine atoms, results in enhanced hydrophobicity.Tanford, C. The Hydrophobic Effect: Formation of Micelles and BiologicalMembranes; 2d ed.; Wiley: New York, 1980. Indeed, partition coefficientspoint to the superior hydrophobicity of CF₃ (Π=1.07) over CH₃ (Π=0.50)groups. Resnati, G. Tetrahedron 1993, 49, 9385-9445. The lowpolarizability of fluorine also results in low cohesive energy densitiesof liquid fluorocarbons and is manifested in their low propensities forintermolecular interactions. Riess, J. G. Colloid Surf.-A 1994, 84,33-48; and Scott, R. L. J. Am. Chem. Soc. 1948, 70, 4090-4093. Theseunique properties of fluorine simultaneously bestow hydrophobic andlipophobic character to biopolymers with high fluorine content. Marsh,E. N. G. Chem. Biol. 2000, 7, R153-R157.

[0003] Introduction of amino acids containing terminal trifluoromethylgroups at appropriate positions on protein folds increases the thermalstability and enhances resistance to chemical denaturants. Bilgicer, B.;Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 4393-4399; and Tang,Y.; Ghirlanda, G.; Vaidehi, N.; Kua, J.; Mainz, D. T.; Goddard, W. A.;DeGrado, W. F.; Tirrell, D. A. Biochemistry 2001, 40, 2790-2796.Furthermore, specific protein-protein interactions can be programmed bythe use of fluorocarbon and hydrocarbon side chains. Bilgicer, B.; Xing,X.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 11815-11816. Becausespecificity is determined by the thermodynamic stability of all possibleprotein-protein interactions, a detailed fundamental understanding ofthe various combinations is essential.

[0004] The so-called “leucine zipper” protein motif, originallydiscovered in DNA-binding proteins but also found in protein-bindingproteins, consists of a set of four or five consecutive leucine residuesrepeated every seven amino acids in the primary sequence of a protein.In a helical configuration, a protein containing a leucine zipper motifpresents a line of leucines on one side of the helix. With two suchhelixes alongside each other, the arrays of leucines can interdigitatelike a zipper and/or form side-to-side contacts, thus forming a stablelink between the two helices. Moreover, an increase in thehydrophobicity of the leucine sidechains, e.g., by substitution ofhydrogens with fluorines, in a leucine zipper motif should increase thestrength of the zipper.

[0005] Selective fluorination of biologically active compounds is oftenaccompanied by dramatic changes in physiological activities. (a) Welch,T.; Eswarakrishnan, S. Fluorine in Bioorganic Chemistry;Wiley-Interscience: New York, 1991 and references cited therein; (b)Fluorine-containing Amino Acids; Kukhar', V. P., Soloshonok, V. A.,Eds.; John Wiley & Sons: Chichester, 1994; (c) Williams, R. M. Synthesisof Optically Active α-Amino Acids, Pergamon Press: Oxford, 1989; (d)Ojima, I.; Kato, K.; Nakahashi, K.; Fuchikami, T.; Fujita, M. J. Org.Chem. 1989, 54, 4511-4522; (e) Tsushima, T.; Kawada, K.; Ishihara, S.;Uchida, N.; Shiratori, O.; Higaki, J.; Hirata, M. Tetrahedron 1988, 44,5375-5387; (f) Weinges, K.; Kromm, E. Liebigs Ann. Chem. 1985, 90-102;(g) Eberle, M. K.; Keese, R.; Stoeckli-Evans, H. Helv. Chim. Acta 1998,81, 182-186; and (h) Tolman, V. Amino Acids 1996, 11, 15-36. Further,fluorinated amino acids have been synthesized and studied as potentialinhibitors of enzymes and as therapeutic agents. Kollonitsch, J.;Patchett A. A.; Marburg, S.; Maycock, A. L.; Perkins, L. M.; Doldouras,G. A.; Duggan, D. E.; Aster, S. D. Nature 1978, 274, 906-908.Trifluoromethyl containing amino acids acting as potentialantimetabolites have also been reported. (a) Walborsky, H. M.; Baum, M.E. J. Am. Chem. Soc. 1958, 80, 187-192; (b) Walborsky, H. M.; Baum, M.;Loncrini, D. F. J. Am. Chem. Soc. 1955, 77, 3637-3640; and (c) Hill, H.M.; Towne, E. B.; Dickey, J. B. J. Am. Chem. Soc. 1950, 72, 3289-3289.

[0006] We describe herein inter alia the design, synthesis,thermodynamic characterization and programmed self-sorting of peptidesystems with orthogonally miscible hydrocarbon and fluorous, i.e.,highly fluorinated cores.

SUMMARY OF THE INVENTION

[0007] A novel, short and efficient synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine (6) in greater than 99% ee startingfrom the protected oxazolidine aldehyde 1 is described. The enantiomericexcess of the product was calculated from an NMR analysis of a dipeptideformed by reaction with a protected L-serine derivative. Furthermore, aracemic sample of N-acylated hexafluoroleucine was enzymaticallyresolved by treatment with porcine kidney Acylase I and was found tohave the same optical rotation as the sample of synthetic 6.

[0008] The invention also relates to a method for efficient resolutionof the four diastereomers of 4,4,4-trifluorovaline and5,5,5-trifluoroleucine. Appropriately derivatized trifluoroamino acidswere separated by flash column chromatography into two enantiomericpairs, which were further resolved by porcine kidney acylase I todeliver four pure diastereomers.

[0009] Another aspect of the present invention relates to theincorporation of hexafluoroleucine as a hydrophobic core residue in adesigned coiled-coil, and tailored highly specific protein-proteininteractions based on the substitution of a hydrophobic core of aprotein with fluorinated residues. Another aspect of the inventionrelates to the design and manipulation of specific helix-helixinteractions within the context of the nonpolar environment ofmembranes.

BRIEF DESCRIPTION OF THE FIGURES

[0010]FIG. 1 depicts helical wheel representation of residues 1-30 of Hand F looking down the superhelical axis from the N-terminus. All sevencore leucines in H were replaced by hexafluoroleucine (L) in F.

[0011]FIG. 2 depicts HPLC traces establishing preferential homodimerformation by fluorous and hydrocarbon cores. Preformed disulfide bondedheterodimer HF (20 μM) was incubated in redox buffer (125 μM oxidizedglutathione, 500 μM reduced glutathione, pH 7.50, 100 mM NaCl, 200 mMMOPS). After 200 minutes, only homodimers and mixed disulfides remain.The mixed heterodimer is estimated to be less than 2% of all H— andF-containing peptides at equilibrium. Peaks marked “*1” and “*2” are Hmonomer and F-glutathione mixed disulfide, respectively, and the peakmarked “**” is an impurity. The equilibrium lies firmly in favor of thehomodimers HH and FF. The free energy of specificity for formation ofhomodimers, ΔG_(spec)=−2.1 kcal/mol.

[0012]FIG. 3 depicts: [A] circular dichroism spectra of HH (◯) and FF() (conditions: [HH]=[FF]=2 μM, pH 7.40, 137 mM NaCl, 2.7 mM KCl, 10 mMPBS, 10° C.); and [B] thermal denaturation profiles of HH (◯), FF ()and HF (♦) (conditions: [HH]=[FF]=[HF]=2 μM, 5 M Gdn HCl, pH 7.40, 137mM NaCl, 2.7 mM KCl, 10 mM PBS).

[0013]FIG. 4 depicts a representative sedimentation equilibrium tracefor FF from analytical ultracentrifugation (conditions: 15 μM peptide(FF) conc., pH 7.40, 10 mM phosphate (pH 7.40), 137 mM NaCl, 2.7 mM KCl.Centrifugation: 26 000 rpm, 18 hours equilibration time, 10° C.MW_(calc) (for dimer of FF)=18132, MW_(found)=17385).

[0014]FIG. 5 depicts a MALDI mass spectrum of purified peptide HH(calcd. =7556.8 [M+], found=7561).

[0015]FIG. 6 depicts a MALDI mass spectrum of purified peptide FF(calcd. =9066 [M+], found=9076.3).

[0016]FIG. 7 depicts a MALDI mass spectrum of purified peptide HF(calcd. =8310.4 [M⁺], found=8317). The smaller peaks in the spectrum arethe M²⁺ peak (4159.9), and monomeric H (3783.3) and monomeric F (4537.7)peptides, resulting from the cleavage of the HF disulfide bond duringthe MALDI experiment.

[0017]FIG. 8 depicts the synthesis of the homodimer and the heterodimer.[A] Disulfide bonded homodimers HH and FF were synthesized by airoxidation of monomeric peptides in 6 M Gdn HCl. [B] The heterodimer HFwas synthesized by reaction of H with Ellman's reagent (ER), followed byreaction with excess F.

[0018]FIG. 9 depicts thermal melting curves for the two homodimers. [A]HH with increasing concentrations of guanidine hydrochloride; and [B] FFmonitored by the decrease in molar ellipticity at 222 nm. Peptideconcentration=2 μM

[0019]FIG. 10 depicts guanidine hydrochloride melting curves for the twoheterodimers. [A] HH (at 74° C.); and [B] FF (at 80° C.). The data yieldan apparent free energy of unfolding: ΔG_(HH)=+3.90 kcal/mol andΔG_(FF)=+16.76 kcal/mol.

[0020]FIG. 11 presents graphically the melting temperatures (T_(m)) as afunction of guanidine hydrochloride concentration for HH (◯) and FF ().At all temperatures, the fluorinated peptide is more stable.

[0021]FIG. 12 depicts a method for the optical resolution oftrifluoromethyl amino acids. The racemic mixture is N-acylated withacetic anhydride (90% yield), followed by enzymatic cleavage to yieldthe α-S isomer (99% yield). The stereochemistry at the β(trifluorovaline) and γ(trifluoroleucine) carbons is still unresolved. Amethod for the production of the N-t-Boc-protected amino acid is alsodepicted.

[0022]FIG. 13 depicts stereospecific syntheses of trifluoroleucine andtrifluoronorvaline from L-homoserine (1). An asterisk indicatesunresolved stereochemistry.

[0023]FIG. 14 depicts stereospecific syntheses of t-Boc-protectedtrifluorovaline, trifluoroisoleucine and hexafluoroleucine fromoxazolidine aldehyde 10, derived from D-serine. [O]=PCC, NaOAc, 4 Åmolecular sieves (yields range from 50-80%); [**] TsOH, MeOH, rt.

[0024]FIG. 15 depicts separation of diastereomeric alcohols 16 by flashcolumn chromatography, followed by oxidation to give enantiomericallypure trifluorovalines. Comparison of the ¹H and ¹⁹F NMR spectra ofdipeptide 21 to the corresponding dipeptide obtained from a mixture of(2S,3S)- and (2S,3R)-trifluorovaline shows that there has been nodetectable racemization of 21.

DETAILED DESCRIPTION OF THE INVENTION

[0025] General Synthesis of Trifluoromethyl Analogs of Leucine,Isoleucine, Valine and Norvaline

[0026] We have invented methods to synthesize t-Boc protectedtrifluoroleucine, trifluorovaline, trifluoroisoleucine,hexafluoroleucine, and trifluoronorvaline, e.g., with α-Sstereochemistry. Xing, X.; Fichera, A.; Kumar, K. “A novel synthesis ofenantiomerically pure 5,5,5,5′,5′,5′-hexafluoroleucine.” Org. Lett.2001, 3, 1285-1286. These are derived from L-homoserine (FIG. 13) orD-serine (FIG. 14). An efficient synthesis of trifluoromethionine hasbeen disclosed. Dannley, R. L.; Taborsky, R. G. “Synthesis ofDL-S-trifluoromethylhomocysteine (trifluoromethylmethionine).” J. Org.Chem. 1957, 10, 1275-76; and Duewel, H.; Daub, E.; Robinson, V.; Honek,J. F. “Incorporation of trifluoromethionine into a phage lysozyrne:Implications and a new marker for use in protein F-19 NMR.” Biochemistry1997, 36, 3404-3416.

[0027] Suitably protected L-homoserine was oxidized to the correspondingaldehyde 2 followed by the fluoride-induced transfer of trifluoromethylgroup from (trifluoromethyl)trimethylsilane. The resulting secondaryfluoro alcohol 3 was oxidized with PCC in 89% yield and then subjectedto Wittig olefination and catalytic hydrogenation to yieldBoc-5,5,5-α-S-trifluorolecuine (6). The stereochemistry at theγ-position is 60% S and 40% R after the reduction reaction, and >99.5% Sat the C_(α)position (ratios were determined by chiral HPLC). Thediastereomerically pure compounds are obtained by reduction to thealcohol followed by chromatographic separation. Alcohol 3 wasdeoxygenated via homolytic reductive cleavage of its thionocarbonateintermediate (7), followed by catalytic reduction to remove the benzylprotecting functionality to yield Boc-5,5,5-α-S-trifluoronorvaline (8).

[0028] To install fluorinated side chains on other amino acids that areusually found in hydrophobic cores, we started with the oxazolidinealdehyde 10 (Garner aldehyde), available from D-serine in four steps(FIG. 14) which serves as a chiral non racemic synthon. Campbell, A. D.;Raynham, T. M.; Taylor, R. J. K. “A simplified route to the (R)-Garneraldehyde and (S)-vinyl glycinol.” Synthesis 1998, 1707-1709; Garner, P.;Park, J. M. “The Synthesis and Configurational Stability ofDifferentially Protected Beta-Hydroxy-Alpha-Amino Aldehydes.” J. Org.Chem. 1987, 52, 2361-2364; and Angrick, M. “Note On the Preparation ofN-Substituted Aminoglyceraldehydes.” Mon. Chem. 1985, 116, 645-649. Atthis stage, trifluoromethyl and pentafluoroethyl groups were introducedusing methodology similar to that described earlier. The secondaryalcohols were then oxidized to the corresponding ketones in good yieldusing PCC. The trifluoromethyl ketone 14 was further subjected to aWittig olefination to yield alkene 15, which after catalytichydrogenation and oxidation gave Boc-4,4,4-α-S-trifluorovaline (17). Thepentafluoroethyl ketone 18 can be subjected to olefination under similarconditions followed by hydrogenation and oxidation to deliverBoc-5,5,5-trifluoroisoleucine (20). Aldehyde 10 is directly convertedinto the hexafluoro olefin 12 using the phosphonium analog ofMiddleton's phosphorane generated in situ fromtetrakis(trifluoromethyl)-1,3-dithietane and triphenylphosphine.Catalytic hydrogenation was then used to unmask the alcohol andsimultaneously reduce the alkene. The resulting alcohol was thenoxidized using PCC to yield Boc-5,5,5,5′,5′,5′-α-S-hexafluoroleucine.

[0029] While the C_(α)stereochemistry is rigorously maintainedthroughout our synthetic scheme, the amino acids produced in this mannerare still a mixture of isomers at the β-position in the case oftrifluorovaline and trifluoroisoleucine. We have found that normal phasechromatography of alcohols 16 and 19 results in clean separation intothe (2S,3S) and (2S,3R) components with recoveries in the 95-100% range.Furthermore, under standard peptide coupling conditions, thestereochemical integrity of the alpha carbon is not compromised.

[0030] We have also taken advantage of enzymatic resolution of racemicamino acids with Acylase I and Lipase. Chenault, H. K.; Dahmer, J.;Whitesides, G. M. “Kinetic resolution of unnatural and rarely occurringamino acids: enantioselective hydrolysis of N-acyl amino acids catalyzedby acylase I.” J. Am. Chem. Soc. 1989, 111, 6354-64; and Houng, J.-Y.;Wu, M.-L.; Chen, S.-T. “Kinetic resolution of amino acid esterscatalyzed by lipases.” Chirality 1996, 8, 418-422. Commerciallyavailable 5,5,5-trifluoroleucine and 4,4,4-trifluorovaline wereacetylated with acetic anhydride and resolved (Acylase I) to yield theα-S amino acids (and >99.9% S stereochemistry at C_(α)) in >90% yield.See FIG. 12; Tsushima, T.; Kawada, K.; Ishihara, S.; Uchida, N.;Shiratori, O.; Higaki, J.; Hirata, M. “Fluorine-containing amino acidsand their derivatives. 7. Synthesis and antitumor activity of α- andγ-substituted methotrexate analogs.” Tetrahedron 1988, 44, 5375; Lazar,J.; Sheppard, W. A. “Fluorinated analogs of leucine, methionine, andvaline.” J. Med. Chem. 1968, 11, 138; Watanabe, H.; Hashizume, Y.;Uneyama, K. “Homologation of trifluoroacetimidoyl iodides bypalladium-catalyzed carbonylation. An approach to α-aminoperfluoroalkanoic acids.” Tetrahedron Lett. 1992, 33, 4333; Larsson, U.;Carlson, R.; Leroy, J. “Synthesis of amino acids with modified principalproperties. 1. Amino acids with fluorinated side chains.” Acta Chem.Scand. 1993, 47, 380-90; Ojima, I.; Kato, K.; Nakahashi, K.; Fuchikami,T.; Fujita, M. “New and effective routes to fluoro analogs of aliphaticand aromatic amino acids.” J. Org. Chem. 1989, 54, 4511-22; Tolmann, V.“Syntheses of fluorinated amino acids. From the classical to the modernconcept.” Amino Acids 1996, 11, 15; Zhang, C.; Ludin, C.; Eberle, M. K.;Stoeckli-Evans, H.; Keese, R. “Asymmetric synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine.” Helv. Chim. Acta 1998, 81, 174;Eberle, M. K.; Keese, R.; Stoeckli-Evans, H. “New synthesis andchirality of (−)-4,4,4,4′,4′,4′-hexafluorovaline.” Helv. Chim. Acta 199881, 182; Keese, R.; Hinderling, C. “Efficient synthesis of (S)-methylhexafluorovalinate.” Synthesis 1996, 695; and Weinges, K.; Kromm, E.“Nonproteinogenic amino acids, II. Synthesis and determination of theabsolute configuration of (2S,4S)-(−)- and(2S,4R)-(+)-5,5,5-trifluoroleucine.” Liebigs Ann. Chem. 1985, 90-102.The selectivity of the acylase reaction was determined by chiral HPLC(CROWNPAK(+)-CR column, Daicel Chemical Industries). The trifluoroderivatives were further Boc protected under mild conditions withoutracemization for use in solid phase peptide synthesis (SPPS).Stereochemistry at the carbon (trifluorovaline) and γ-carbon(trifluoroleucine) was left unresolved. In contrast, racemichexafluorovaline resisted resolution by either Lipase or Acylase I.

[0031] Specific Synthesis of (S)-5,5,5,5,5′, 5′-Hexafluoroleucine

[0032] A novel, short and efficient synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine (6) in greater than 99% ee startingfrom the protected oxazolidine aldehyde 1 is described. The enantiomericexcess of the product was calculated from an NMR analysis of a dipeptideformed by reaction with a protected L-serine derivative. Furthermore, aracemic sample of N-acylated hexafluoroleucine was enzymaticallyresolved by treatment with porcine kidney Acylase I and was found tohave the same optical rotation as the sample of synthetic 6.

[0033] Herein, we disclose a novel and efficient synthesis of(S)-5,5,5,5′,5′,5′-hexafluoroleucine starting from commericallyavailable D-serine. For synthesis of α-amino acids derived from D-serineusing a serine aldehyde equivalent, see: Blaskovich, M. A.; Lajoie, G.A. J. Am. Chem. Soc. 1993, 115, 5021-5030. While there is one existingreport of the synthesis of racemic hexafluoroleucine (Lazar, J.;Sheppard, W. A. J. Med. Chem. 1968, 11, 138), and another recent reportdetailing the preparation of 6 in 81% ee (Zhang, C.; Ludin, C.; Eberle,M. K.; Stoeckli-Evans, H.; Keese, R. Helv. Chim. Acta 1998, 81,174-181), we have discovered a method to obtain hexafluoroleucine in>99%ee, e.g., for direct use in solid phase peptide synthesis.

[0034] Our synthesis commenced from the oxazolidine aldehyde 1 (Garneraldehyde) which served as a chiral, nonracemic synthon. See (a) Garner,P.; Park, J. M. J. Org. Chem. 1987, 52, 2361-2364. (b) Garner, P.; Park,J. M. J. Org. Chem. 1988, 53, 2979-2984. (c) Garner, P.; Park, J. M.;Malecki, E. J. Org. Chem. 1988, 53, 4395-4398. (d) Angrick, M. Montash.Chem. 1985, 116, 645-649. Aldehyde 1 is derived from D-serine and wasobtained using a slight modification of a published procedure and isexceptionally stable towards racemization in subsequent steps. Campbell,A. D.; Raynham, T. M.; Taylor, R. J. K. Synthesis 1998, 1707-1709. In akey step, aldehyde 1 was converted to the bis-trifluoromethyl olefin 2by a Wittig reaction in 92% yield (Scheme 1). See Korhummel, C.; Hanack,M. Chem. Ber. 1989, 122, 2187-2192.

[0035] Scheme 1. Reagents and conditions: (a) PPh₃, [(CF₃)₂C]₂S₂, Et₂O,−78° C. →rt, 3 d, 92%; (b) H₂, 10% Pd/C, THF, 98%; (c) TsOH, MeOH, rt, 1d, 80%; (d) PDC, DMF, 18 hrs., 75%; (e) 40% CF₃CO₂H/CH₂Cl₂; HCl wash, 10min., rt, >95%.

[0036] The ylide for this reaction is the phosphonium analog ofMiddleton's phosphorane, Middleton, W. J.; Sharkey, W. H. J. Org. Chem.1965, 30, 1384, generated in situ fromtetrakis(trifluoromethyl)-1,3-dithietane (Anello, L. G.; Vanderpuy, M.J. Org. Chem. 1982, 47, 377-378), and triphenyl phosphine. See (a)Burton, D. J.; Yang, Z. Y.; Qiu, W. M. Chem. Rev. 1996, 96, 1641-1715.(b) Dixon, D. A.; Smart, B. E. J. Am. Chem. Soc. 1986, 108, 7172-7177.(c) Burton, D. J.; Inouye, Y. Tetrahedron Lett. 1979, 3397-3400; and (d)Kobayashi, Y.; Nakajima, M.; Nakazawa, M.; Taguchi, T.; Ikekawa, N.;Sai, H.; Tanaka, Y.; Deluca, H. F. Chem. Pharm. Bull. 1988, 36,4144-4147. The olefin 2 was reduced by catalytic hydrogenation over Pd/Cto give the suitably substituted oxazolidine 3 in 98% yield. Next, theoxazolidine was subjected to acid catalyzed ring cleavage unmasking thealcohol 4. Alcohol 4 was oxidized to the carboxylic acid 5 usingpyridinium dichromate and in the final step, the t-butyloxycarbonylgroup was removed using trifluoroacetic acid to yield the hydrochloridesalt of the desired α-amino acid 6. While the last deprotection step wascarried out in order to verify the optical purity of 6, the Bocprotected amino acid 5 could be directly used for solid phase synthesisof peptides.

[0037] The optical purity of synthetic 6 was verified in two ways. Aracemic sample of 5 (prepared using a different route) and 5 obtainedthrough the scheme described here were separately coupled to a protectedmethyl ester of L-serine (7), and the resulting dipeptide was analyzedusing ¹H NMR spectroscopy.

[0038] In the case of the dipeptide obtained from racemic 5, threesignals corresponding to the t-Boc group, the methyl ester and thet-butyl ether were split into two peaks, presumably due to formation oftwo diastereomers; whereas, 5 from the present synthesis yielded adipeptide with only one set of signals for the same three sets ofprotons. Further, racemic 6 was N-acylated and enzymatically resolvedusing porcine kidney Acylase I [E.C.N. 3.5.1.14] to yield the α-S isomerexclusively. See (a) Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J.Am. Chem. Soc. 1989, 111, 6354-64; and (b) Fu, S. C. J.; Birnbaum, S. M.J. Am. Chem. Soc. 1953, 75, 918-920. The optical rotation of 6 obtainedin this manner and that of the synthetic sample were identical. Thus,the synthesis proceeds in greater than >99% ee. The NMR data for 6 agreewith those reported previously. Moreover, both the synthetic sample andthe enzyme resolved samples of 6 had [α]²⁶⁰ _(D)=+5.6° (c 1, CH₃OH).Likewise, the construction of 5,5,5,5′,5′,5′-(R)-hexafluoroleucine wasachieved from L-senne.

[0039] Resolution of the Diastereomers of 4,4,4-Trifluorovaline and5,5,5-Trifluoroleucine

[0040] Reported here is an efficient resolution of the fourdiastereomers of 4,4,4-trifluorovaline (TFV) and 5,5,5-trifluoroleucine(TFL). The method as outlined in Scheme 1 is simple and practical.Appropriately derivatized TFL and TFV could be separated into twoenantiomeric pairs by flash column chromatography. Subsequent enzymaticdeacylation of the N-acetyl enantiomeric pairs of amino acids withporcine kidney acylase I delivers all four diastereomers in opticallypure form. Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am. Chem.Soc. 1989, 111, 6354-6364.

[0041] In the course of our study on the synthesis of enantiomericallypure TFV, we found that Boc-protected 4,4,4-trifluorovalinol α-Sdiastereomers could be easily separated by column chromatography onsilica gel. This finding encouraged us to develop a resolution schemefor racemic TFV and TFL. As shown in Scheme 2, Boc-TFV 1 was firstconverted to Boc-trifluorovalinol 2 via esterification of 1 with methyliodide, followed by reduction of the methyl ester with sodiumborohydride in methanol in 73% overall yield for the two steps. Theracemic mixture of trifluorovalinols was easily separated into the twoenantiomeric pairs 2a [(2S,3R)+(2R,3S)] and 2b [(2S,3S)+(2R,3R)] bycolumn chromatography on silica gel using n-pentane/ethyl ether (1:1) aseluant. Although the methyl esters of Boc-TFV 1 are also separable, theyare not stable toward racemization in the subsequent reduction step.Oxidation of the hydroxyl group of 2a and 2b with PDC in DMF, removal ofthe Boc-protecting group with 30% trifluoroacetic acid in methylenechloride followed by acylation of the free amino group afforded theN-acetyl amino acids 3a and 3b respectively. Finally, enzymaticdeacylation of 3a and 3b with porcine kidney acylase I afforded the fourdiastereomers 4a-d. Only those diastereomers that had an S configurationat C_(α)were deacylated by the enzyme. Removal of the acetyl group fromthe two C_(α)-R diastereomers was realized by refluxing with 3 N HCl.

[0042] Reagents and conditions: (a) NaHCO₃, CH₃I, DMF, rt, 95%; (b)NaBH₄, CH₃OH, 77%; flash column chromatography, n-pentane/Et₂O (1:1),silica gel:2 (300:1); (c) PDC, DMF, rt, 65%; (d) 30% CF₃CO₂H/CH₂Cl₂,100%; (e) NaOH/H₂O, Ac₂O, 0° C., 95%; (f) Porcine kidney acylase I, pH7.50, 25° C., 95%; (g) 3N HCl, 98%.

[0043] This strategy was also applied to the resolution of TFL (Scheme3). Initially, Boc-TFL 5 was also converted to the correspondingalcohols following the procedure used for Boc-TFV 1, but we found thatthe trifluoroleucinols were not separable by column chromatography onsilica gel. Interestingly, the methyl esters of 5 were readily separatedinto two pairs 6a and 6b on silica gel using n-pentane/ethyl ether (3:1)as eluant and were stable toward racemization in the reduction step. TheN-acetyl amino acids 7a and 7b were obtained from 6a and 6b respectivelyby straightforward functional group transformations, which includedreduction of the methyl ester group to hydroxyl, oxidation of thehydroxyl to acid, and replacement of the Boc-protecting group with anacetyl group. In the final step, enzymatic deacylation was applied to 7aand 7b to give diastereomerically pure compounds 8a-d.

[0044] Reagents and conditions: (a) NaHCO₃, CH₃I, DMF, rt, 95%; flashcolumn chromatography, n-pentane/Et₂O (4:1), silica gel:6 (400:1); (b)NaBH4, CH₃OH, 94%; (c) PDC, DMF, rt, 60%; (d) 30% CF₃CO₂H/CH₂Cl₂, 100%;(e) NaOH/H₂O, Ac₂O, 0° C., 95%; (f) Porcine kidney acylase I, pH 7.5,25° C., 95%; (g) 3N HCl, 96%.

[0045] The purity of the intermediates and the final diastereomers wasascertained using ¹H, ¹³C and ¹⁹F NMR spectroscopy. The ¹⁹F NMRtechnique is particularly useful in this case for purity control due toits high sensitivity and the large chemical shift dispersion observedfor these compounds. The enantiomeric pairs exhibited baseline separated¹⁹F NMR spectra in each case. Contamination by the other enantiomericpair or racemization during chemical transformation could be easilydetected. The optical purity of the products was also verified by NMRanalysis of dipeptides formed by coupling with a side chain protectedmethyl ester of L-serine. Xing, X.; Fichera, A.; Kumar, K. Org. Lett.2001, 3, 1285-1286. The ¹⁹F NMR spectra clearly showed four peaks fordipeptides derived from the racemic mixture, two peaks for dipeptidesderived from enantiomeric pairs, and only one peak for thediastereomerically pure dipeptide.

[0046] Programmed Sel-Sorting of Coiled Coils with Leucine andHexafluoroleucine Cores

[0047] The coiled coil motif offers an excellent model system to explorespecificity in protein-protein interactions. Lupas, A. Curr. Opin.Struct. Biol. 1997, 7, 388-393; and Lupas, A. Trends Biochem.Sci. 1996,21, 375-382. These protein interaction motifs represent small,synthetically tractable targets for testing hypothetical constructs.Lajmi, A. R.; Lovrencic, M. E.; Wallace, T. R.; Thomlinson, R. R.; Shin,J. A. J. Am. Chem. Soc. 2000, 122, 5638-5639. The α-helical coiled coilis typically composed of a number of parallel or antiparallel α-heliceswrapped around one another with a shallow left-handed superhelicaltwist. Crick, F. H. C. Acta Crystallographica 1953, 6, 689-697. Theycontain a heptad repeat, whose positions are denoted a-g, where the aand d positions are hydrophobic residues that form the interface betweenhelices, and constitute the primary driving force for oligomerization.Additionally, interhelical electrostatic interactions between e and gresidues provide a secondary source of stability. Monera, 0. D.; Zhou,N. E.; Kay, C. M.; Hodges, R. S. J. Biol. Chem. 1993, 268, 19218-19227;and Monera, 0. D.; Kay, C. M.; Hodges, R. S. Biochemistry 1994, 33,3862-3871. From the crystal structures of 32-residue synthetic coiledcoils, it is estimated that nearly 900 Å² surface area per helix isburied at a dimeric interface and nearly 1640 Å² per helix in atetramer. Harbury, P. B.; Zhang, T.; Kim, P. S.; Alber, T. Science 1993,262, 1401-1407; and O'Shea, E. K.; Klemm, J. D.; Kim, P. S.; Alber, T.Science 1991, 254, 539-544. The importance of hydrophobic surface areafor coiled coil stability has been extensively studied through the useof de novo designed synthetic peptide models. Zhu, B. Y.; Zhou, N. E.;Kay, C. M.; Hodges, R. S. Protein Sci. 1993, 2, 383-394; and Zhu, B. Y.;Zhou, N. E.; Semchuk, P. D.; Kay, C. M.; Hodges, R. S. Int. J. Pept.Protein Res. 1992, 40, 171-179. These interaction surfaces are thereforeideally suited to study the effect of fluorination on the driving forceand specificity.

[0048] Peptides were synthesized by the in situ neutralization protocolfor t-Boc synthesis on 0.40 mmol NH₂ eq g⁻¹ methylbenzhydrylamine (MBHA)resin. At the end of linear synthesis, the formyl protecting group onthe tryptophan residue was removed by treatment with 1:10 piperidine inDMF solution at 0° C. for 2 hrs. Further treatment with anhydrous HFresulted in the simultaneous removal of all side-chain protecting groupsand cleavage of the peptide chain from the resin. The peptides werepurified on reversed-phase HPLC using a linear gradient of acetonitrilein 0.1% trifluoroacetic acid (TFA)/water. The analytical purity of thepeptides was confirmed by HPLC, amino acid analysis and MALDI massspectrometry.

[0049] The disulfide bonded dimers of H(HH), F (FF) and the mixed dimerHF were synthesized by two different methods. The homodimers HH and FFwere synthesized by overnight air oxidation of the monomeric peptides in6 M guanidine hydrochloride (Gdn HCl) at pH 8.50 (50 mM Tris). Theheterodimer HF was synthesized by reaction of H with a large excess ofEllman's reagent (ER, CAS No. 69-78-3) to produce an activated disulfidespecies at pH 7.50, followed by reaction with excess monomeric F at pH5.10. Riddles, P. W.; Blakeley, R. L.; Zemer, B. Methods Enzymol. 1983,91, 49-60. The resulting heterodimer HF was purified by reversed-phaseHPLC.

[0050] Peptides H and F are equipped with N-terminal cysteine residuesand were designed to form parallel homodimeric coiled coil assemblies.Wolf, E.; Kim, P. S.; Berger, B. Protein Sci. 1997, 6, 1179-1189. Thesepeptides have an identical sequence except that all seven of the coreleucine residues in H have been replaced by5,5,5,5′,5′,5′-α-S-hexafluoroleucine in F, shielding 28 trifluoromethylgroups from aqueous solvent in the canonical fluorinated dimer. FIG. 1.Hexafluoroleucine was synthesized according to the procedure describedherein. Xing, X.; Fichera, A.; Kumar, K. Org. Lett. 2001, 3, 1285-1286.The peptides were assembled on 4-methylbenzhydrylamine (MBHA) resinaccording to the in situ neutralization protocol for t-Boc peptidesynthesis, as described previously, and purified by reverse-phase HPLC.Schnolzer, M.; Alewood, P.; Jones, A.; Alewood, D.; Kent, S. B. Int. J.Pept. Protein Res. 1992, 40, 180-193. Purity of the peptides wasconfirmed by analytical HPLC and MALDI mass spectrometry. H and F aredesigned to form parallel coiled coil structures due to unfavorableinterhelical electrostatic interactions in the antiparallelarrangements. See Lumb, K. J.; Kim, P. S. Biochemistry 1995, 34,8642-8648; and Harbury, P. B.; Zhang, T.; Kim, P. S.; Alber, T. Science1993, 262, 1401-1407. Furthermore, a single polar residue, Asn14, whichcan only hydrogen bond in the parallel arrangement, was incorporated inthe hydrophobic core. See Oakley, M. G.; Kim, P. S. Biochemistry 1998,37, 12603-12610; and McClain, D. L.; Woods, H. L.; Oakley, M. G. J. Am.Chem. Soc. 2001, 123, 3151-3152. The peptides were equipped with aGly-Gly-Cys tripeptide at the NH₂-terminus. The cysteine residue permitsredox chemistry in the form of disulfide-thiol equilibrium, and the twoglycine residues provide a flexible linker. Disulfide bonded dimers ofH(HH) and F (FF) were synthesized by air oxidation of the monomericpeptides in pH 8.50 Tris buffer.

[0051] The extent of the preference for sorting into homodimericpopulations under equilibrium conditions was examined by a disulfideexchange assay. See Harbury, P. B.; Kim, P. S.; Alber, T. Nature 1994,371, 80-83; Oakley, M. G.; Kim, P. S. Biochemistry 1998, 37,12603-12610; and Saghatelian, A.; Yokobayashi, Y.; Soltani, K.; Ghadiri,M. R. Nature 2001, 409, 797-80. Preformed disulfide bonded heterodimerHF was incubated in a pH 7.50 redox buffer at 20° C., conditions underwhich disulfide exchange is rapid. Aliquots were removed from thereaction at various times and quenched with 5% trifluoroacetic acid. Thetime points were then analyzed by analytical reversed-phase HPLC.Relative concentrations of the disulfide bonded hetero- and homodimerswere estimated by integration of the area under corresponding peaks at230 nm. Within 30 minutes of the start of the reaction, the heterodimerdisproportionates into the two homodimers HH and FF. Specifically, weobserved about 10% of the H-gluathione and 20% of the F-glutathionedisulfide adducts. Coincidentally, the H-glutathione disulfide co-elutedwith HH. After 200 minutes, only a trace of the heterodimer (˜3%)remains. FIG. 2. Further change in the reaction mixture was not observedeven after 18 hours. Assuming that the glycyl linkers allow thecysteines to exchange randomly under redox buffer conditions, the dataindicate that the homodimers are preferred over the heterodimer by26-fold. In order to establish that the reaction had reachedequilibrium, we placed an equimolar amount of the reduced peptides H andF under similar redox buffer conditions, and monitored the reaction for18 hours. Again, the heterodimer accounted for only 3% of all disulfidebonded species. Unambiguous stepwise synthesis of the heterodimer HFconfirms that the disulfide bond forming chemistry is reversible andunder thermodynamic control, and that there are no kinetic barriers tothe formation of the disulfide bonded heterodimer HF. The heterodimer HFwas synthesized by reaction of H with Ellman's reagent to produce anactivated disulfide species. This mixed disulfide was then reacted withexcess monomeric F to yield HF. See Riddles, P. W.; Blakeley, R. L.;Zemer, B. Methods Enzymol. 1983, 91, 49-60.

[0052] Accordingly, peptides H and F are predisposed to form homodimers.See Otto, S.; Furlan, R. L. E.; Sanders, J. K. M. J. Am. Chem. Soc.2000, 122, 12063-12064; Hioki, H.; Still, W. C. J. Org. Chem. 1998, 63,904-905; and Rowan, S. J.; Hamilton, D. G.; Brady, P. A.; Sanders, J. K.M. J. Am. Chem. Soc. 1997, 119, 2578-2579. The relative instability ofthe heterodimer and the hyperstability of the fluorinated dimer providethe driving force for preferential homodimer formation. From the peakratios at equilibrium, the free energy of specificity for the formationof homodimers, ΔG_(spec), is calculated to be at least −2.1 kcaumol. SeeExample 11. TABLE 1 Melting temperatures and solution MWs for disulfidebonded dimers. Peptide T_(m) (° C.)^(a) MW_(app) (no. of helices)^(c) HH34  7501 ± 38 (2) HF 36  8815 ± 63^(d) (2) FF 82 (45^(b)) 17835 ± 75 (4)

[0053] Circular dichroism spectra of peptides HH, HF and FF revealed thealpha helical character of all three disulfide bonded dimers, showingcharacteristic minima at 208 and 222 nm. FIG. 3[A]. The order ofstability was readily established when melting curves were monitored byCD are compared. All three peptides HF, RH and FF displayed cooperativeunfolding transitions as a function of temperature in the presence ofguanidine hydrochloride (Gdn HCl). The melting temperatures in 5 M GdnHCl of HH (34° C.) and that of HF (36° C.) were similar. In contrast,the fluorinated peptide FF meltrd at an estimated 82° C. under theseconditions. FIG. 3[B]. The fluorinated disulfide bonded dimer displayedremarkable stability, resisting even minimal denaturation at 6 M Gdn HClat room temperature. Even at 7 M Gdn HCl concentration, FF resistedthermal denaturation up to 45° C. Table 1. Thus, the fluorinatedassembly FF is significantly more stable than either the heterodimer HFor the hydrocarbon homodimer HH. A priori, the T_(m) of the heterodimercan be expected to be the average of the T_(m) values of the homodimers(ΔT_(m)=0). The specificity for heterodimer formation can beapproximated by ΔT_(m)=T_(m)(heterodimer HF) −½ [T_(m)(homodimerHH)+T_(m)(homodimer FF)]=−22° C. Differences in ΔT_(m) have been invokedto explain the specificity of the heterodimeric Fos-Jun peptide pair.O'Shea, E. K.; Rutkowski, R.; Kim, P. S. Cell 1992, 68, 69-708. In ourcase, ΔT_(m) is −22° C., i.e. the thermal stability of the heterodimeris appreciably lower than the expected intermediate stability. Thethermodynamic consequence of the relative stability of the fluorinatedpeptide assembly FF and the instability of HF is to shift theequilibrium away from the heterodimer to the homodimers.

[0054] Sedimentation equilibrium analysis of the disulfide bonded dimersin the 2-15 μM range revealed that HH has an apparent molecular weightof 7501 D in solution, consistent with two helices forming the coiledcoil structure. Table 1. In contrast, FF sediments with an apparentmolecular weight of 17835 D. This could be due to much largerassociation constant of FF monomers or due to the larger size of thecore formed by hexafluoroleucine forcing it to adopt a coiled coilstructure with four helices.

[0055] In sum, we have demonstrated the incorporation ofhexafluoroleucine as the sole hydrophobic core residue in a designedcoiled-coil. Furthermore, this is the first example of a very highlyspecific protein-protein interaction based on the substitution of thehydrophobic core with fluorinated residues. This aspect of the inventionrelates to a method to design and manipulate specific helix-helixinteractions within the context of the nonpolar environment ofmembranes. See Choma, C.; Gratkowski, H.; Lear, J. D.; DeGrado, W. F.Nature Struct. Biol. 2000, 7, 161-166; and Zhou, F. X.; Cocco, M. J.;Russ, W. P.; Brunger, A. T.; Engelman, D. M. Nature Struct. Biol. 2000,7, 154-160.

[0056] Definitions

[0057] For convenience, certain terms employed in the specification,examples, and appended claims are collected here.

[0058] The term “heteroatom” as used herein means an atom of any elementother than carbon or hydrogen. Preferred heteroatoms are boron,nitrogen, oxygen, phosphorus, sulfur and selenium.

[0059] The term “electron-withdrawing group” is recognized in the art,and denotes the tendency of a substituent to attract valence electronsfrom neighboring atoms, i.e., the substituent is electronegative withrespect to neighboring atoms. A quantification of the level ofelectron-withdrawing capability is given by the Hammett sigma (a)constant. This well known constant is described in many references, forinstance, J. March, Advanced Organic Chemistry, McGraw Hill BookCompany, New York, (1977 edition) pp. 251-259. The Hammett constantvalues are generally negative for electron donating groups (σ[P]-0.66for NH2) and positive for electron withdrawing groups (σ[P]=0.78 for anitro group), σ[P] indicating para substitution. Exemplaryelectron-withdrawing groups include nitro, acyl, formyl, sulfonyl,trifluoromethyl, cyano, chloride, and the like. Exemplaryelectron-donating groups include amino, methoxy, and the like.

[0060] The term “alkyl” refers to the radical of saturated aliphaticgroups, including straight-chain alkyl groups, branched-chain alkylgroups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkylgroups, and cycloalkyl substituted alkyl groups. In preferredembodiments, a straight chain or branched chain alkyl has 30 or fewercarbon atoms in its backbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀for branched chain), and more preferably 20 or fewer. Likewise,preferred cycloalkyls have from 3-10 carbon atoms in their ringstructure, and more preferably have 5, 6 or 7 carbons in the ringstructure.

[0061] Unless the number of carbons is otherwise specified, “loweralkyl” as used herein means an alkyl group, as defined above, but havingfrom one to ten carbons, more preferably from one to six carbon atoms inits backbone structure. Likewise, “lower alkenyl” and “lower alkynyl”have similar chain lengths. Preferred alkyl groups are lower alkyls. Inpreferred embodiments, a substituent designated herein as alkyl is alower alkyl.

[0062] The term “aralkyl”, as used herein, refers to an alkyl groupsubstituted with an aryl group (e.g., an aromatic or heteroaromaticgroup).

[0063] The terms “alkenyl” and “alkynyl” refer to unsaturated aliphaticgroups analogous in length and possible substitution to the alkylsdescribed above, but that contain at least one double or triple bondrespectively.

[0064] The term “aryl” as used herein includes 5-, 6- and 7-memberedsingle-ring aromatic groups that may include from zero to fourheteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole,oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazineand pyrimidine, and the like. Those aryl groups having heteroatoms inthe ring structure may also be referred to as “aryl heterocycles” or“heteroaromatics.” The aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl,alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,sulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, or the like. The term “aryl” alsoincludes polycyclic ring systems having two or more cyclic rings inwhich two or more carbons are common to two adjoining rings (the ringsare “fused rings”) wherein at least one of the rings is aromatic, e.g.,the other cyclic rings can be cycloalkyls, cycloalkenyls, cycloalkynyls,aryls and/or heterocyclyls.

[0065] The terms ortho, meta andpara apply to 1,2-, 1,3- and1,4-disubstituted benzenes, respectively. For example, the names1,2-dimethylbenzene and ortho-dimethylbenzene are synonymous.

[0066] The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, more preferably 3- to 7-membered rings,whose ring structures include one to four heteroatoms. Heterocycles canalso be polycycles. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, quinoline,phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline,pteridine, carbazole, carboline, phenanthridine, acridine, pyrimidine,phenanthroline, phenazine, phenarsazine, phenothiazine, furazan,phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine,piperazine, morpholine, lactones, lactams such as azetidinones andpyrrolidinones, sultams, sultones, and the like. The heterocyclic ringcan be substituted at one or more positions with such substituents asdescribed above, as for example, halogen, alkyl, aralkyl, alkenyl,alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido,phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio,sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic orheteroaromatic moiety, —CF₃, —CN, or the like.

[0067] The terms “polycyclyl” or “polycyclic group” refer to two or morerings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle can be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl,ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromaticmoiety, —CF₃, —CN, or the like.

[0068] As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

[0069] The terms “amine” and “amino” are art-recognized and refer toboth unsubstituted and substituted amines, e.g., a moiety that can berepresented by the general formula:

[0070] wherein R₉, R₁₀ and R′₁₀ each independently represent a grouppermitted by the rules of valence.

[0071] The term “acylamino” is art-recognized and refers to a moietythat can be represented by the general formula:

[0072] wherein R₉ is as defined above, and R′₁₁ represents a hydrogen,an alkyl, an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as definedabove.

[0073] The term “amido” is art recognized as an amino-substitutedcarbonyl and includes a moiety that can be represented by the generalformula:

[0074] wherein R₉, R₁₀ are as defined above. Preferred embodiments ofthe amide will not include imides which may be unstable.

[0075] The term “alkylthio” refers to an alkyl group, as defined above,having a sulfur radical attached thereto. In preferred embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R₈, wherein m and R₈ are defined above.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

[0076] The term “carbonyl” is art recognized and includes such moietiesas can be represented by the general formula:

[0077] wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R₈ or apharmaceutically acceptable salt, R′ 11 represents a hydrogen, an alkyl,an alkenyl or —(CH₂)_(m)—R₈, where m and R₈ are as defined above. WhereX is an oxygen and R₁₁ or R′₁₁ is not hydrogen, the formula representsan “ester”. Where X is an oxygen, and R₁₁ is as defined above, themoiety is referred to herein as a carboxyl group, and particularly whenR₁₁ is a hydrogen, the formula represents a “carboxylic acid”. Where Xis an oxygen, and R′ ₁₁ is hydrogen, the formula represents a “formate”.In general, where the oxygen atom of the above formula is replaced bysulfur, the formula represents a “thiolcarbonyl” group. Where X is asulfur and R₁₁ or R′₁₁ is not hydrogen, the formula represents a“thiolester.” Where X is a sulfur and R₁₁ is hydrogen, the formularepresents a “thiolcarboxylic acid.” Where X is a sulfur and R₁₁′ ishydrogen, the formula represents a “thiolformate.” On the other hand,where X is a bond, and R₁₁ is not hydrogen, the above formula representsa “ketone” group. Where X is a bond, and R₁₁ is hydrogen, the aboveformula represents an “aldehyde” group.

[0078] The terms “alkoxyl” or “alkoxy” as used herein refers to an alkylgroup, as defined above, having an oxygen radical attached thereto.Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy and the like. An “ether” is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of an alkyl thatrenders that alkyl an ether is or resembles an alkoxyl, such as can berepresented by one of —O-alkyl, —O-alkenyl, —O-alkynl, —O—(CH₂)_(m)—R₈,where m and R₈ are described above.

[0079] The term “sulfonate” is art recognized and includes a moi ty thatcan be represented by the general formula:

[0080] in which R₄₁ is an electron pair, hydrogen, alkyl, cycloalkyl, oraryl.

[0081] The terms triflyl, tosyl, mesyl, and nonaflyl are art-recognizedand refer to trifluoromethanesulfonyl, p-toluenesulfonyl,methanesulfonyl, and nonafluorobutanesulfonyl groups, respectively. Theterms triflate, tosylate, mesylate, and nonaflate are art-recognized andrefer to trifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

[0082] The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

[0083] The term “sulfate” is art recognized and includes a moiety thatcan be represented by the general formula:

[0084] in which R₄₁ is as defined above.

[0085] The term “sulfonylamino” is art recognized and includes a moietythat can be represented by the general formula:

[0086] The term “sulfamoyl” is art-recognized and includes a moiety thatcan be represented by the general formula:

[0087] The term “sulfonyl”, as used herein, refers to a moiety that canbe represented by the general formula:

[0088] in which R₄₄ is selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.

[0089] The term “sulfoxido” as used herein, refers to a moiety that canbe represented by the general formula:

[0090] in which R₄₄ is selected from the group consisting of hydrogen,alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

[0091] A “selenoalkyl” refers to an alkyl group having a substitutedseleno group attached thereto. Exemplary “selenoethers” which may besubstituted on the alkyl are selected from one of —Se-alkyl,—Se-alkenyl, —Se-alkynyl, and —Se—(CH₂)_(m)—R₇, m and R₇ being definedabove.

[0092] Analogous substitutions can be made to alkenyl and alkynyl groupsto produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls,amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls,carbonyl-substituted alkenyls or alkynyls.

[0093] As used herein, the definition of each expression, e.g. alkyl, m,n, etc., when it occurs more than once in any structure, is intended tobe independent of its definition elsewhere in the same structure.

[0094] The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts,P.G.M. Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: NewYork, 1991).

[0095] Certain compounds of the present invention may exist inparticular geometric or stereoisomeric forms. The present inventioncontemplates all such compounds, including cis- and trans-isomers, R-and S-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

[0096] If, for instance, a particular enantiomer of a compound of thepresent invention is desired, it may be prepared by asymmetricsynthesis, or by derivation with a chiral auxiliary, where the resultingdiastereomeric mixture is separated and the auxiliary group cleaved toprovide the pure desired enantiomers. Alternatively, where the moleculecontains a basic functional group, such as amino, or an acidicfunctional group, such as carboxyl, diastereomeric salts are formed withan appropriate optically-active acid or base, followed by resolution ofthe diastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

[0097] For purposes of this invention, the chemical elements areidentified in accordance with the Periodic Table of the Elements, CASversion, Handbook of Chemistry and Physics, 67th Ed., 1986-87, insidecover.

COMPOUNDS OF THE INVENTION

[0098] In certain embodiments, the present invention relates to acompound represented by A:

[0099] wherein

[0100] X represents O, S, N(R), or C(R)₂;

[0101] R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

[0102] R′ represents H, alkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; or XR′ represents halide;

[0103] the stereochemical configuration at any stereocenter of acompound represented by A may be R, S, or a mixture of theseconfigurations; and

[0104] the enantiomeric excess of a compound represented by A is greaterthan or equal to about 85%.

[0105] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R).

[0106] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

[0107] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H.

[0108] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein R′ represents H, alkyl, or aralkyl.

[0109] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein R′ represents H.

[0110] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

[0111] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0112] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

[0113] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); and R′ represents H, alkyl, or aralkyl.

[0114] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

[0115] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.

[0116] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.

[0117] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

[0118] In certain embodiments, the compounds of the present inventionare represented by general structure A and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

[0119] In certain embodiments, the present invention relates to acompound represented by B:

[0120] wherein

[0121] X represents O, S, N(R), or C(R)₂;

[0122] R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

[0123] R′ represents H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; or XR′ represents halide;

[0124] the stereochemical configuration at any stereocenter of acompound represented by B may be R, S, or a mixture of theseconfigurations; and

[0125] the enantiomeric excess of a compound represented by B is greaterthan or equal to about 85%.

[0126] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R).

[0127] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

[0128] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H.

[0129] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.

[0130] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein R′ represents H.

[0131] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

[0132] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0133] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

[0134] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); and R′ represents H, aralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

[0135] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

[0136] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H,aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0137] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

[0138] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

[0139] In certain embodiments, the compounds of the present inventionare represented by general structure B and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

[0140] In certain embodiments, the present invention relates to acompound represented by C:

[0141] wherein

[0142] X represents O, S, N(R), or C(R)₂;

[0143] R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

[0144] R′ represents H, alkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; or XR′ represents halide;

[0145] the stereochemical configuration at any stereocenter of acompound represented by C may be R, S, or a mixture of theseconfigurations; and

[0146] the enantiomeric excess of a compound represented by C is greaterthan or equal to about 85%.

[0147] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R).

[0148] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

[0149] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein R represents independently for each occurrence H.

[0150] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein R′ represents H, alkyl, or aralkyl.

[0151] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein R′ represents H.

[0152] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

[0153] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0154] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

[0155] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); and R′ represents H, alkyl, or aralkyl.

[0156] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

[0157] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.

[0158] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.

[0159] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

[0160] In certain embodiments, the compounds of the present inventionare represented by general structure C and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

[0161] In certain embodiments, the present invention relates to acompound represented by

[0162] wherein

[0163] X represents O, S, N(R), or C(R)₂;

[0164] R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

[0165] R′ represents H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; or XR′ represents halide;

[0166] the stereochemical configuration at any stereocenter of acompound represented by D may be R, S, or a mixture of theseconfigurations; and

[0167] the enantiomeric excess of a compound represented by D is greaterthan or equal to about 85%.

[0168] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R).

[0169] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

[0170] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein R′ represents independently for each occurrence H.

[0171] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.

[0172] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein R′ represents H.

[0173] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

[0174] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0175] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

[0176] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); and R′ represents H, aralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

[0177] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

[0178] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H,aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0179] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

[0180] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

[0181] In certain embodiments, the compounds of the present inventionare represented by general structure D and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

[0182] In certain embodiments, the present invention relates to acompound represented by E:

[0183] wherein

[0184] X represents O, S, N(R), or C(R)₂;

[0185] R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

[0186] R′ represents H, alkyl, aryl, heteroaryl, aralkyl, orheteroaralkyl; or XR′ represents halide;

[0187] the stereochemical configuration at any stereocenter of acompound represented by E may be R, S, or a mixture of theseconfigurations; and

[0188] the enantiomeric excess of a compound represented by E is greaterthan or equal to about 85%.

[0189] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R).

[0190] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

[0191] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein R represents independently for each occurrence H.

[0192] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein R′ represents H, alkyl, or aralkyl.

[0193] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein R′ represents H.

[0194] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

[0195] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0196] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

[0197] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); and R′ represents H, alkyl, or aralkyl.

[0198] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

[0199] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.

[0200] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, alkyl, or aralkyl.

[0201] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

[0202] In certain embodiments, the compounds of the present inventionare represented by general structure E and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

[0203] In certain embodiments, the present invention relates to acompound represented by F:

[0204] wherein

[0205] X represents O, S, N(R), or C(R)₂;

[0206] R represents independently for each occurrence H, alkyl, aryl,heteroaryl, aralkyl, heteroaralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl;

[0207] R′ represents H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; or XR′ represents halide;

[0208] the stereochemical configuration at any stereocenter of acompound represented by F may be R, S, or a mixture of theseconfigurations; and

[0209] the enantiomeric excess of a compound represented by F is greaterthan or equal to about 85%.

[0210] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R).

[0211] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl,or aralkylaminocarbonyl.

[0212] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein R represents independently for each occurrence H.

[0213] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.

[0214] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein R′ represents H.

[0215] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein R represents independently for each occurrence H; and R′represents H.

[0216] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0217] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); and R represents independently for eachoccurrence H.

[0218] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); and R′ represents H, aralkyl, formyl,acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

[0219] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); and R′ represents H.

[0220] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H,aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.

[0221] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.

[0222] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H.

[0223] In certain embodiments, the compounds of the present inventionare represented by general structure F and the attendant definitions,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.

[0224] In certain embodiments, the present invention relates to acompound represented by any of the structures outlined above, whereinthe enantiomeric excess of said compound is greater than or equal toabout 90%.

[0225] In certain embodiments, the present invention relates to acompound represented by any of the structures outlined above, whereinthe enantiomeric excess of said compound is greater than or equal toabout 95%.

[0226] In certain embodiments, the present invention relates to acompound represented by any of the structures outlined above, whereinsaid compound is a single stereoisomer.

[0227] In certain embodiments, the present invention relates to acompound represented by any of the structures outlined above, whereinsaid compound is in the form of a salt.

[0228] In certain embodiments, the present invention relates to aformulation, comprising a compound represented by any of the structuresoutlined above; and a pharmaceutically acceptable excipient.

[0229] In certain embodiments, the present invention relates to anoligopeptide or a polypeptide, comprising a compound represented by anyof the structures outlined above.

METHODS OF THE INVENTION

[0230] In certain embodiments, the present invention relates to a methodof resolving into individual enantiomers a mixture of diastereomers of acompound represented by structure A, B, C, D, E, or F, comprising thesteps of:

[0231] (a) using chromatography to obtain an individual pair ofenantiomers of a compound represented by structure A, B, C, D, E, or Ffrom a mixture of diastereomers of said compound; and

[0232] (b) using enzymatic hydrolysis to obtain a single enantiomer ofsaid compound from the individual pair of enantiomers of said compound.

[0233] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers.

[0234] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(tert-butyloxycarbonyl)HN in the mixture of diastereomers.

[0235] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents (acyl)HN inthe the individual pair of enantiomers subjected to enzymatichydrolysis.

[0236] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents (acetyl)HN inthe the individual pair of enantiomers subjected to enzymatichydrolysis.

[0237] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein the enzyme used is porcinekidney acylase I.

[0238] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers; and (R)₂N represents(acyl)HN in the the individual pair of enantiomers subjected toenzymatic hydrolysis.

[0239] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(tert-butyloxycarbonyl)HN in the mixture of diastereomers; and (R)₂Nrepresents (acetyl)HN in the the individual pair of enantiomerssubjected to enzymatic hydrolysis.

[0240] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers; (R)₂N represents(acyl)HN in the the individual pair of enantiomers subjected toenzymatic hydrolysis; and the enzyme used is porcine kidney acylase I.

[0241] In certain embodiments, the present invention relates to theaforementioned resolution method, wherein (R)₂N represents(tert-butyloxycarbonyl)HN in the mixture of diastereomers; (R)₂Nrepresents (acetyl)HN in the the individual pair of enantiomerssubjected to enzymatic hydrolysis; and the enzyme used is porcine kidneyacylase I.

[0242] In certain embodiments, the present invention relates to a methodof synthesizing a non-native oligopeptide, polypeptide or protein withenhanced hydrophobicity relative to a native oligopeptide, polypeptideor protein, comprising the step of using a compound represented bystructure A, B, C, D, E, or F in place of a leucine or valine in asynthesis of an oligopeptide, polypeptide or protein.

[0243] In certain embodiments, the present invention relates to theaforementioned method of synthesizing a non-native oligopeptide,polypeptide or protein with enhanced hydrophobicity, wherein thesynthesis is automated.

[0244] In certain embodiments, the present invention relates to a methodof enhancing the hydrophobicity of an oligopeptide, polypeptide orprotein, comprising the step of replacing a leucine or valine in anoligopeptide, polypeptide or protein with a compound represented bystructure A, B, C, D, E, or F.

[0245] In certain embodiments, the present invention relates to a methodof synthesizing a trifluoromethyl-containing analogue of norvaline orvaline, comprising the steps of:

[0246] (a) oxidizing a protected serine or homoserine to give analdehyde;

[0247] (b) reacting the aldehyde with trimethyl(trifluoromethyl)silaneand fluoride to give a secondary alcohol;

[0248] (c) acylating the secondary alcohol using an arylchlorothionoformate to give a thionocarbonate; and

[0249] (d) reducing the thionocarbonate using a tin hydride and aninitiator to give a trifluoromethyl-containing analogue of norvaline orvaline.

[0250] In certain embodiments, the present invention relates to a methodof synthesizing a trifluoromethyl-containing analogue of leucine,comprising the steps of:

[0251] (a) oxidizing a protected homoserine to give an aldehyde;

[0252] (b) reacting the aldehyde with trimethyl(trifluoromethyl)silaneand fluoride to give a secondary alcohol;

[0253] (c) oxidizing the secondary alcohol to give a trifluoromethylketone;

[0254] (d) reacting the trifluoromethyl ketone with(methylene)triphenylphosphine to give an alkene; and

[0255] (e) hydrogenating the alkene to give a trifluoromethyl-containinganalogue of leucine.

[0256] In certain embodiments, the present invention relates to a methodof synthesizing protected 5,5,5,5′,5′,5′-hexafluoroleucine, comprisingthe steps of:

[0257] (a) reacting an oxazolidine aldehyde derived from serine with ahexafluoroisopropylidene ylide to give an oxazolidine1,1-bis(trifluoromethyl)alkene;

[0258] (b) hydrogenating the oxazolidine 1,1-bis(trifluoromethyl)alkeneto give an oxazolidine 1,1-bis(trifluoromethyl)alkane;

[0259] (c) hydrolyzing the oxazolidine 1,1-bis(trifluoromethyl)alkane togive a protected amino alcohol; and

[0260] (d) oxidizing the protected amino alcohol to give protected5,5,5,5′,5′,5′-hexafluoroleucine.

[0261] In certain embodiments, the present invention relates to theaforemnetioned method of synthesizing protected5,5,5,5′,5′,5′-hexafluoroleucine, wherein the reagents for step (a)comprise triphenylphosphine and [(CF₃)₂C]₂S₂; the reagents for step (b)comprise hydrogen and 10% palladium on carbon; the reagents for step (c)comprise toluenesulfonic acid and methanol; and the reagents for step(d) comprise pyridinium dichromate.

[0262] In certain embodiments, the present invention relates to a methodof preparing a compound represented by 6, comprising the steps depictedin Scheme 1:

[0263] Exemplification

[0264] The invention now being generally described, it will be morereadily understood by reference to the following examples, which areincluded merely for purposes of illustration of certain aspects andembodiments of the present invention, and are not intended to limit theinvention.

EXAMPLE 1

[0265] General Experimental Procedures

[0266] Melting points were determined in open capillaries on a MEL-TEMPII apparatus (Laboratory Devices, Inc., Holliston, Mass.) and areuncorrected. All reactions requiring non-aqueous conditions wereperformed in oven-dried glassware under positive pressure of argon.Flash column chromatography was performed by forced flow of solventusing Kieselgel 60 SiO₂ (230-240 mesh) gel (EM Science) packed intoglass columns using standard litertaure procedures. Still, W. C.; Kahn,M.; Mitra, A. J. Org. Chem. 1978, 43, 2923. Analytical thin layerchromatography was performed using E. Merck silica gel Kieselgel 60 F₂₅₄(0.25 mm) plates. Compounds were visualized by UV light, exposure toiodine vapour or by staining with a ninhydrin solution followed byheating. Reagents and solvents were of reagent grade or better and wereobtained from Aldrich Chemical Co., Fluka Chemie AG, Lancaster Synthesisor Novabiochem Corp. Deuterated solvents were obtained from CambridgeIsotope Laboratories.

[0267] Infra-red spectra were obtained on a Mattson 1000 FT-IRinstrument with a 4 cm⁻1 bandpass. Spectra of solid samples wereobtained as solid thin-films or dissolved in thin layers of organicsolvents between NaCl plates. Mass Spectra were obtained on a HewlettPackard GC-MS (Model 5988A) with a dip-probe using conditions asindicated. Nuclear magnetic resonance spectra were recorded on a BrukerAM-300 or a Bruker DPX-300 instrument in standard deuterated solvents.Optical rotations were measured using an AUTOPOL IV digital polarimeter(Rudolph Research Analytical, N.J.).

EXAMPLE 2

[0268] Synthesis of Bis-trifluoromethyl Olefin (2)

[0269] Typical procedure for the coupling reaction: To a stirredsolution of the Garner aldehyde 1 (7.0 g, 31.0 mmol) and PPh₃ (57 g, 217mmol) in dry Et₂O (300 mL) was added2,2,4,4-tetrakis-(trifluoromethyl)-1,3-dithietane (39.5 g, 108.5 mmol)at −78° C. under argon. The mixture was stirred for 3 d while beingslowly warmed to room temperature. The reaction slowly accumulated aninsoluble white solid which was filtered and the filtrate concentrated.The residue was further dissolved in n-pentane (300 mL) and filteredagain to remove insoluble impurities. After removal of the solvent, theresidue was subjected to flash column chromatography usingn-pentane/Et₂O (6/1) as eluant to give pure 2 as a pale yellow oil (10.4g, 92%). ¹H NMR (300 MHz, CDCl₃) δ 6.70 (d, 1H, J=8.7 Hz), 4.81 (bs,1H), 4.23 (dd, 1H, J=6.9 Hz, 9.3 Hz), 3.79 (dd, 1H, J=3.9 Hz, 9.3 Hz),1.65 (s, 3H), 1.56 (s, 3H), 1.42 (s, 9H); ¹⁹F NMR (282.6 MHz,CDCl₃/CFCl₃) δ −65.01 (d, 3F, J=5.9 Hz), −58.44 (d, 3F, J=5.9 Hz); FT-IR(film, V_(max), cm⁻¹) 2983m, 2935m, 2885w, 1713s, 1479w, 1460w, 1379s,1230s, 1165s, 1110m, 971m; [α]_(D) ^(26.1)=+12.3° (c 1.7, CHCl₃); GC-MS(CI, CH₄): 364 (1, [M+1]⁺), 336 (18), 308 (100), 288 (98), 264 (37), 102(2), 57 (9).

EXAMPLE 3

[0270] Synthesis of Oxazolidine (3)

[0271] A 500 mL round bottomed flask was charged with a solution of 2(10.3 g, 28.3 mmol) in THF (250 mL) and 10% Pd/C (40 g). The reactionflask was purged with argon and hydrogen sequentially and stirred underhydrogen at room temperature until uptake of H₂ ceased (24 hours). Thecatalyst was then separated from the reaction mixture by filtration (andcan be used again). The filtrate was dried over anhydrous MgSO₄ andconcentrated by rotary evaporation to give 3 (10.1 g, 98% yield) as apale yellow oil. ¹H NMR (300 MHz, CDCl₃) δ 4.23 (4.05) (m, 1H), 4.00(dd, 1H, J=5.4 Hz, 9.3 Hz), 3.73 (d, 1H, J=9.3 Hz), 3.58 (3.05) (m, 1H),2.18 (2.01) (m, 2H), 1.62 (1.58) (s, 3H), 1.48 (br. s, 12H); ¹³C NMR(75.5 MHz, CDCl₃) δ 153.22 (151.51) (C═O), 123.89 (q, 2×CF₃,¹J_(CF)=284.0), 94.47 (94.03) (C), 80.85 (80.73) (C), 67.26 (66.65)(CH₂), 55.58 (55.12) (CH), 45.44 (45.12) (quintet, CH, ²J_(CF)=27.2 Hz),28.98 (28.00) (CH₂), 28.25 (3×CH₃), 27.58 (26.90) (CH₃), 24.15 (22.86)(CH₃); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ−67.68-−68.42 (m); FT-IR (film,v_(max), cm⁻¹): 2984m, 2941m, 2884w, 1704s, 1457m, 1393s, 1258s, 1168s,1104s, 847m; [α]_(D) ^(22.4)=+17.5° (c 0.4, CHCl₃); GC-MS (Cl, CH₄): 366(4, [M+1]+), 338 (16), 310 (100), 290 (48), 266 (48), 57 (8).

EXAMPLE 4

[0272] Synthesis of N-Boc-5,5,5,5′,5′,5′-(S)-Hexafluoroleucinol (4)

[0273] To a solution of3 (10.1 g, 27.6 mmol) in CH₂Cl₂ (30 mL) was added10 mL of trifluoroacetic acid (TFA). The reaction mixture was stirred atroom temperature for 5 min. After removal of the solvent and TFA, theresidue was partitioned between 150 mL of ethyl ether and 100 mL of H₂O.The organic layer was washed with water (20 mL×4), dried over MgSO₄, andconcentrated to give 4 (7.2 g, 80% yield) as a white solid. The aqueouslayers contain a completely deprotected product due to cleavage of theBOC moiety as evidenced by ninhydrin active material. Thishexafluoroamino alcohol can be converted back to 4 by protecting thefree amine group as a BOC amide. ¹H NMR (300 MHz, CDCl₃) δ 5.03 (d, 1H,J=8.1 Hz), 3.84 (m, 1H), 3.70 (m, 2H), 3.20 (m, 1H), 3.10 (br. s, 1H),1.98 (m, 2H), 1.45 (s, 9H); ¹³C NMR (75.5 MHz, CDCl₃) δ 156.57 (C═O),124.00 (q, 2×CF₃, ¹J_(CF)=284.0 Hz), 80.58 (C), 66.08 (CH₂), 50.57 (CH),45.09 (m, CH, ²J_(CF)=28.1 Hz), 28.38 (3×CH₃), 26.44 (CH₂); ¹⁹F NMR(282.6 MHz, CDCl₃/CFCl₃) δ −67.96 (m), −68.46 (m); FT-IR (KBr pellet,v_(max), cm⁻¹) 3397s (br), 3253s, 3068m, 2981s, 2948m, 1686s, 1552s,1369s, 1289s, 1174s, 1145s, 1055s; [α]_(D) ^(22.9)=−14.4° (c 1.0,CH₃OH); GC-MS (Cl, CH₄): 326 (8, [M+1]⁺), 298 (14), 270 (100), 226 (20),57 (2); m.p.=114-115° C.

EXAMPLE 5

[0274] Synthesis of N-Boc-5,5,5,5′,5′,5′-(S)-Hexafluoroleucine (5)

[0275] A mixture of 4 (7.1 g, 21.8 mmol) and pyridinium dichromate (33g, 88 mmol) in DMF (150 mL) was stirred under argon at room temperaturefor 24 hrs. before 150 mL of H₂O was added. The mixture was thenextracted with ethyl ether (400 mL×2). The combined ether layers werewashed with 1 N HCl (80 mL×2) and concentrated until about 150 mL ofsolution left. This solution was washed with 5% NaHCO₃ (150 mL×3). Thecombined aqueous layers were acidified to pH 2 with 3 N HCl, extractedwith ether again (400 mL×2). The ether layers were then dried over MgSO₄and concentrated to give 5 (5.2 g, 70%) as a white solid. ¹H NMR (300MHz, CDCl₃) δ 7.36 (5.21) (d, 1H, J=6.3 Hz), 4.41 (m, 1H), 3.37 (m, 1H),2.43-2.11 (br. m, 2H), 1.47 (s, 9H); ⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ−67.87-−68.23 (m); FT-IR (KBr pellet, v_(max), cm⁻¹) 3358-2500m (br.),3245s, 3107m, 2989s, 2980m, 1725s, 1712s, 1657s, 1477s, 1458s, 1404s,1296s, 1277s, 1258s, 916m; [α]_(D) ^(21.8)=−23.0° (c 1.0, CH₃OH); GC-MS(CI, CH₄): 340(21, [M+1]+), 312 (7), 284 (100), 264 (16), 240 (19), 57(39); m.p.=85-91° C.

EXAMPLE 6

[0276] Synthesis of 5,5,5,5′,5′,5′-(S)-Hexafluoroleucine (6)

[0277] A solution of 5 (581 mg, 1.7 mmol) in 5 mL of TFA/CH₂Cl₂ (2/3)was stirred for 30 min. After removal of the solvents, the residue waspartitioned between 1 N HCl (10 mL×3) and ethyl ether (10 mL). Thecombined aqueous layers were freeze dried to give 6 (446 mg, 95% yield)as a white solid.

EXAMPLE 7

[0278] Synthesis of Dipeptide (8)

[0279] To a stirred solution of 5 (11 mg, 0.03 mmol) in anhydrous DMF (1mL) was added diisopropyl ethyl amine (13 mg, 0.1 mmol), HBTU (13 mg,0.03 mmol), and H-Ser(t-Bu)—OMe HCl (14 mg, 0.065 mmol) sequentially.The mixture was stirred at room temperature for 40 min before 6 mL ofH₂O was added. The reaction mixture was extracted with ether (15 ml) andthe organic layer was futher washed with 1 N HCl (5 mL×2) and 5% NaHCO₃solution (5 ml), dried over MgSO₄, and concentrated to afford 8 (13 mg,87% yield) as a white solid. ¹H NMR (300 MHz, CDCl₃) δ 6.68 (d, 1H,J=8.1 Hz), 5.21 (d, 1H, J=8.1 Hz), 4.64 (m, 1H), 4.40 (m, 1H), 3.86 (dd,1H, J=2.7 Hz, 9.3 Hz), 3.76 (s, 3H), 3.56 (dd, 1H, J=3.3 Hz, 9.3 Hz),3.50 (m, 1H), 2.33-2.10 (br. m, 2H), 1.45 (s, 9H), 1.14 (s, 9H).

EXAMPLE 8

[0280] Incorporation of 5,5,5,5′,5′,5′,5′-hexafluoroleucine intoPeptides

[0281] The incorporation of hexafluoroleucine in a 30-residue peptidewith the sequence given below has been achieved. Leucines in bold are5,5,5,5′,5′,5′-(S)-hexafluoroleucine.

[0282] Peptide 1: Ac-NH-AQLKKELQALKKENAQLKWELQALKKELAQCONH2

[0283] The MALDI-MS of purified peptide 1 (Calc. 4316.8, found 4317.1)confirms the purity and identity of the peptide. Circular dichroism dataindicates that the peptide can readily adopt an alpha helical secondarystructure (characterisitic minima at 208 and 222 nm). Furtherbiophysical studies with these peptides are in progress.

[0284] Peptide Synthesis

[0285] Peptides were prepared using the N-tert-butyloxycarbonyl (t-Boc)amino acid derivatives for Merrifield manual solid-phase synthesis (MBHAresin) using the in-situ neutralization/HBTU protocol on a 0.2 mmolscale. Schnolzer, M.; Alewood, P.; Jones, A.; Alewood.; D, Kent, S. B.Int. J. Pept. Protein Res. 1992, 40, 180-193. N-α-Boc-α-S-amino acidswere used with the following side chain protecting groups: Arg(Tos),Asp(OBzl), Asn(Xan), Gln(Xan), Glu(OBzl) and Lys(2-CI-Z). Peptidecoupling reactions were carried out with 4-fold excess (0.8 mmol) ofactivated amino acid for at least 15 min. Peptides were cleaved from theresin using high HF conditions (90% anhydrous HF/0% anisole at 0° C. for1.5 hours) with simultaneous removal of the side chain protectinggroups. Tam J. P.; Merrifield, R. B. In The Peptides; Udenfriend, S.,Meienhofer, J. Eds.; Academic Press Inc.: New York, 1987; Vol. 9, p 185.

[0286] In the case of hexafluoroleucine, the coupling time was extendedto 2 hrs. The extent of reaction was verified by a Kaiser test aftereach coupling. The N-terminal was acetylated by treatment with 1:4acetic anhydride/DMF and 6 eq. of diisopropylethylamine. The formylprotecting group on the tryptophan residue was removed by treating theresin with 1:10 piperidine in DMF solution. Peptides were cleaved fromthe resin by using high HF conditions (90% anhydrous HF/10% anisole at0° C. for 1.5 h). Crude peptides were extracted with 25% acetic acid andlyophilized. Freeze dried material was dissolved in 0.1% TFA, desaltedand purified by reversed phase HPLC [Vydac C4 column with a 30 minlinear gradient of acetonitrile/H₂O/0.1% TFA at 8.0 mL/min].

[0287] HH: An aqueous solution of H (10 mg, 2.64 μmol) in 50 mM Tris (pH8.50) and 6 M Gdn HCl (total volume: 0.75 mL) was stirred overnight atroom temperature. The reaction was quenched by addition of 250 μLglacial acetic acid and diluted with 1 mL water. The mixture wasdirectly purified by reversed phase HPLC. The fractions containing HHwere collected and lyophilized to deliver 9.0 mg (90%) of HH. MALDI-MS:MW_(calcd)=7556.8, found: 7561.

[0288] FF: An aqueous solution of F (14 mg, 3.09 μmol) in 50 mM Tris (pH8.50) and 6.5 M Gdn HCl (total volume: 1 mL) was stirred overnight atroom temperature. The reaction was quenched by addition of 300 μLglacial acetic acid and diluted with 1.5 mL water. The mixture wasdirectly purified by reversed phase HPLC. The fractions containing FFwere pooled and lyophilized to deliver 12.1 mg (86%) of FF. MALDI-MS:MW_(calcd)=9066, found: 9076.3.

[0289] HF: To an aqueous solution of H (8 mg, 2.11 μmol) in MOPS buffer(pH 7.50) was added 5,5′-dithiobis(2-nitrobenzoic acid) (20 mg, 50.4μmol). The reaction was stirred for 15 minutes and then quenched by theaddition of 300 μL of neat TFA. The reaction mixture was then extractedwith Et₂O (4×10 mL). The aqueous layer was then directly injected into areversed-phase C18 column and purified. The fractions containing themixed disulfide of the Ellman's reagent and H were combined andlyophilized to obtain 8.4 mg of the desired product (95%). The mixeddisulfide (8 mg, 1.92 μmol) was dissolved in a pH 1.50 solutioncontaining F (17.4 mg, 3.84 μmol). The pH was carefully adjusted to 5.10by sequential addition of 0.1 N NaOH solution. The reaction was allowedto proceed for 20 minutes and then quenched by addition of 300 μL TFA.The reaction mixture was then directly purified by reversed phase HPLCto obtain 10 mg of pure HF (62.6%). Nearly 25% of the starting mixeddisulfide was recovered unreacted. MALDI-MS: MW_(calcd): 8310.4, found:8317.

[0290] Purification

[0291] Peptides were desalted and purified by reversed phase HPLC [VydacC₄ column using a 30 min linear gradient of 34-47% acetonitrile/H₂O/0.1%TFA at 8.0 mL/min]. Peptide 1 eluted at ˜43.2% acetonitrile/H₂O/0.1% TFA(˜30.0 min. elution time).

EXAMPLE 9

[0292] Circular Dichroism

[0293] Circular dichroism spectra were obtained on a JASCO J-715spectropolarimeter fitted with a PTC-423S single position Peltiertemperature controller. Buffer conditions were usually 10 mM phosphate(pH 7.40), 137 mM NaCl, 2.7 mM KCl unless otherwise noted. Thespectrometer was calibrated with an aqueous solution of recrystallizedd₁₀-(+)-camphorsulfonic acid at 290.5 nm. The concentrations of thepeptide stock solutions were determined by amino-acid analysis or bymeasuring tryptophan absorbance in 6 M Gdn HCl (assuming an extinctioncoefficient of 5600 M⁻cm⁻1 at 281 nm). Edelhoch, H. Biochemistry 1967,6, 1948. Mean residue ellipticities (deg cm² dmol⁻1) were calculatedusing the relation:

[θ]=θ_(obs)×MRW/10lc  (1)

[0294] wherein θ_(obs); is the measured signal (ellipticity) inmillidegrees, l is the optical pathlength of the cell in cm, c isconcentration of the peptide in mg/mL and MRW is the mean residuemolecular weight (molecular weight of the peptide divided by the numberof residues).

[0295] Thermal denaturation studies were carried out at theconcentrations indicated by monitoring the change in [θ]₂₂₂ as afunction of temperature. Temperature was increased in steps of 0.5° C.with an intervening equilibration time of 120s. Data was collected over16 s per point. The T_(m) was determined from the minima of the firstderivative of [θ]₂₂₂ with T⁻1, where Tis in K.

EXAMPLE 10

[0296] Analytical Ultracentrifugation

[0297] Apparent molecular masses were determined by sedimentationequilibrium on a Beckman XL-A ultracentrifuge. Loading peptideconcentrations were 2-15 μM in 10 mM phosphate (pH 7.40), 137 mM NaCl,2.7 mM KCl. The samples were centrifuged at 32 000 and 26 000 rpm for 18hours at 10° C. before absorbance scans were performed.

[0298] Data obtained at 10° C. were fit globally to the followingequation (2) that describes the sedimentation of a homogeneous species:

Abs=A′ exp (H×M[x ² −x ₀ ²])+B  (2)

[0299] wherein Abs=absorbance at radius x, A′=absorbance at referenceradius x₀, H=(1−{overscore (V)}p)ω²/2RT, {overscore (V)}=partialspecific volume=0.758 mL/g, ρ=density of solvent=1.0017 g/mL, ω=angularvelocity in radians/sec, and M=apparent molecular weight, B=solventabsorbance (blank). We estimated partial specific volume using aminoacid composition (Cohn, E. J., Edsall, J. T. Proteins, Amino Acids andPeptides as Ions and Dipolar Ions. New York, Reinhold, 1943)substituting leucine for hexafluoroleucine in the case of HF and FF forlack of available data.

EXAMPLE 11

[0300] Calculation of Free Energies

[0301] The thermodynamic cycle used for calculating ΔG_(spec) (freeenergy of specificity for the formation of homodimers) is depictedbelow. The superscripts U and F refer to the unfolded and folded statesrespectively of the disulfide bonded dimeric peptides.

[0302] K_(redox) is the equilibrium constant for the redox reaction.K_(random) is the equilibrium constant for the chance pairing of FF, HHand HF peptides and is assumed to be 2 as there are two equivalent waysfor the formation of the heterodimer HF but only one way to form eachhomodimer. O'Shea, E. K.; Rutkowski, R.; Kim, P. S. Cell 1992, 68,69-708. FFFF^(F) is the dimer of the disulfide bonded dimer FF andK_(tetramer) is the equilibrium constant for it's formation. K_(redox)was estimated from equilibrium ratios of HH, FF and HF.

$K_{redox} = \frac{\left\lbrack {HF}^{F} \right\rbrack}{{\left\lbrack {FF}^{F} \right\rbrack^{0.5}\left\lbrack {HH}^{F} \right\rbrack}^{0.5}}$

 ΔG_(spec)(for homodimers)=−{ΔG_(redox)+RT In 2}

EXAMPLE 12

[0303] Calculation of Free Energy of Unfolding for Homomdimer

[0304] The free energy of unfolding for HH was determined by assuming atwo state equilibrium between folded and unfolded states:

[0305] where FHH is the folded species and UHH represents the fullyunfolded HH. Data were obtained by monitoring [θ]₂₂₂ as a function ofGdn HCl concentration. Data were analyzed by the linear extrapolationmethod to yield the free energy of unfolding. The equilibrium constantand therefore ΔG^(o) are easily determined from the average fraction ofunfolding. Assuming that the linear dependence of ΔG^(o) with denaturantconcentration in the transition region continues to zero concentration,the data can be extrapolated to obtain ΔG_(h) ₂ _(O) ^(o), the freeenergy difference in the absence of denaturant. Pace, C. N. Methods inEnzymol. 1995, 259, 538-554; and Tanford, C. Adv.Protein Chem. 1962, 17,69-165.

[0306] Sedimentation equilibrium experiments suggest FF is a tetramer(dimer of the disulfide bonded dimer) in the 2-15 μM concentrationrange. Therefore, we used a unfolded monomer-folded dimer equilibrium tocalculate ΔG° of unfolding:

[0307] where K_(d)=[UFF]²/[F_(FF)] (U_(FF)=unfolded FF and F_(FF)=foldeddimer of FF with 4 helices). Since the total peptide concentrationP_(t), can be given by P_(t)=2 [F_(FF)]+[U_(FF)], the observed CD signalY_(obs) can be described in terms of folded and unfolded baselines,Y_(fol) and Y_(unfol), respectively by the following expression.$\begin{matrix}{Y_{obs} = {{\left( {Y_{unfol} - Y_{fol}} \right)\frac{\sqrt{K_{d}^{2} + {8K_{d}P_{1}}} - K_{d}}{4P_{1}}} + Y_{fol}}} & (2)\end{matrix}$

[0308] Additionally, K_(d) can be expressed in terms of the free energyof unfolding.

K_(d)=exp (−ΔG^(o)/RT)  (3)

[0309] Assuming that the apparent free energy difference between foldedF_(FF) and unfolded U_(FF) states is linearly dependent on the Gdn HClconcentration, ΔG^(o) can be written as:

ΔG^(o)=ΔG_(H) ₂ _(O) ^(o) −m[Gdn.HCl]  (4)

[0310] where ΔG_(H) ₂ _(O) ^(o) is the free energy difference in theabsence of denaturant and m is the dependency of the unfoldingtransition with respect to the concentration of Gdn HCl. The data wasfit for two parameters, ΔG_(H) ₂ _(O) ^(o), and m by nonlinear leastsquares fitting.

EXAMPLE 13

[0311] N-Boc-4,4,4-trifluorovalinol (2)

[0312] To a suspension of Boc-DL-trifluorovaline (1.30 g, 4.79 mmol) andNaHCO₃ (1.21 g, 14.37 mmol) in 20 mL of dry DMF was added 0.33 mL ofCH₃I (5.27 mmol) at room temperature under argon. The resulting mixturewas stirred for 5 h and then partitioned between 75 mL of ethyl acetateand 50 mL of water. The organic layer was washed with water (3×50 mL),dried over MgSO₄, and concentrated to yield 1.36 g (95%) of theBoc-DL-trifluorovaline methyl ester as a pale-yellow oil.

[0313] The Boc-TFV methyl ester (855 mg, 3 mmol) was dissolved in 20 mLof methanol, and NaBH4 (681 mg, 18 mmol) was added in small portions at0° C. The reaction mixture was stirred overnight at room temperature andthen diluted with 80 mL of ethyl acetate, washed with water (3×50 mL),and dried over MgSO₄. After removal of the solvent, the crude product(Boc-trifluorovalinol) was chromatographed on a silica gel column(silica gel, 300 g) using n-pentane/Et₂O (1:1) as eluant to give 452 mgof 2a as a pale-yellow solid (58%) and 214 mg of 2b as a white solid(28%). (2S,3R)-, (2R, 3S)-N-Boc-4,4,4-trifluorovalinol (2a)

[0314]¹H NMR (300 MHz, CDCl₃) δ 5.04 (d, 1H, J=9.3 Hz), 4.02 (m, 1H),3.62 (m, 3H), 2.61 (m, 1H), 1.44 (s, 9H), 1.15 (d, 3H, J=7.2 Hz); ¹³CNMR (75.5 MHz, CDCl₃) δ 156.20 (C═O), 127.83 (q, CF₃, ¹J_(CF)=279.9 Hz),80.26 (C), 62.78 (CH₂), 51.09 (CH), 38.47 (q, CH, ²J_(CF)=25.6 Hz), 28.40 (3×CH₃), 8.76 (CH₃); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −70.63 (d,3F, J=9.0 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3435s, 3300s, 2990s,2979m, 2954m, 1691s, 1539s, 1537s, 1265s, 1172s, 1125; GC-MS (Cl, CH₄):258 (14, [M+1]⁺), 242 (4), 202 (100), 158 (37), 57 (14).

[0315] (2S, 3S)- , (2R, 3R)-N-Boc-4,4,4-trifluorovalinol (2b)

[0316]¹H NMR (300 MHz, CDCl₃) δ 5.11 (d, 1H, J=8.4 Hz), 3.80 (m, 1H),3.66 (m, 2H), 3.45 (t, 1H, J=5.7 Hz), 2.53 (m, 1H), 1.42 (s, 9H), 1.15(d, 3H, J=7.2 Hz); ¹³C NMR (75.5 MHz, CDCl₃) δ 156.43 (C═O), 127.91 (q,CF₃, ¹J_(CF)=280.2 Hz), 80.30 (C), 62.92 (CH₂), 52.56 (CH), 38.89 (q,CH, ²J_(CF=)24.8 Hz), 28. 40 (3 ÅCH₃), 10.59 (CH₃); ¹⁹F NMR (282.6 MHz,CDCl₃/CFCl₃) δ −68.76 (d, 3F, J=8.5 Hz); FT-IR (film, v_(max) cm⁻¹):3436s, 3302s, 3012m, 2990m, 2954m, 1691s, 1532s, 1265s, 1172s, 1127s;GC-MS (Cl, CH₄): 258 (14, [M+1]⁺), 242 (4), 202 (100), 182 (8), 57 (14).

EXAMPLE 14

[0317] (2S,3R)-, (2R,3S)-N-Ac-4.4,4-trifluorovaline (3a)

[0318] A solution of alcohol 2a (257 mg, 1 mmol) in 4 mL of dry DMF wastreated with PDC (2.26 g, 6 mmol) at room temperature under argon andstirred overnight. The reaction mixture was then diluted with 20 mL ofdiethyl ether/30 mL of saturated NaHCO₃ solution. The organic layer waswashed with 10 mL of saturated NaHCO₃. The combined aqueous layers wereacidified to pH 2 with 3 N HCl and extracted with diethyl ether (2×50mL). The combined organic layers were dried over MgSO₄ and concentratedto yield 176 mg of the corresponding Boc-trifluorovaline (65%).

[0319] Boc-TFV (176 mg, 0.65 mmol) was treated with 4 mL of 40%trifluoroacetic acid in CH₂Cl₂ for 10 min. After removal of the solvent,the residue was dissolved in 2 mL of water, treated with NaOH (260 mg,6.5 mmol) at 0° C., followed by dropwise addition of acetic anhydride(0.13 mL, 1.3 mmol). The reaction mixture was stirred at 0° C. for 30min before it was allowed to warm to room temperature. After stirringfor another 1.5 h, the mixture was diluted with 10 mL of water,acidified to pH 2 with 1 N HCl, and extracted with ethyl acetate (2×60mL). The combined organic layers were dried over MgSO₄ and concentratedto give the desired product 3a as a white solid (132 mg, 95%). ¹H NMR(300 MHz, D₂O) δ 4.96 (d, 1H, J=3.0 Hz), 3.07 (m, 1H), 2.04 (s, 3H),1.15 (d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −71.63 (d,3F, J=8.8 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3397s (br), 3253s,3068m, 2981s, 2948m, 1686s, 1552s, 1369s, 1289s, 1174s, 1145s, 1055s;GC-MS (CI, CH₄): 214 (100, [M+1]+), 196 (9), 172 (33), 82 (33), 57 (6).

[0320] (2S,3S)-, (2R,3R)-N-Ac-4,4,4-trifluorovaline (3b)

[0321]¹H NMR (300 MHz, D₂O) δ 4.67 (d, 1H, J=3.3 Hz), 3.07 (m, 1H), 2.04(s, 3H), 1.17 (d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ−69.43 (d, 3F, J=8.8 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3397s (br),3253s, 3068m, 2981s, 2948m, 1686s, 1552s, 1369s, 1289s, 1174s, 1145s,1055s; GC-MS (CI, CH₄): 214 (100, [M+1]+), 196 (9), 172 (33), 101 (10),82 (33), 57 (6).

EXAMPLE 15

[0322] (2S,3R)-4.4.4-Trifluorovaline (4a)

[0323] To a solution of 3a (107 mg, 0.5 mmol) in 1 mL of pH 7.9 aq.LiOH/HOAc was added porcine kidney acylase I (10 mg) at 25° C. Themixture was stirred at 25° C. for 48 h (pH was maintained at 7.5 byperiodic addition of 1 N LiOH). The reaction was then diluted with 5 mLof water, acidified to pH 5.0, heated to 60° C. with Norit, andfiltered. The filtrate was acidified to pH 1.5 and extracted with ethylacetate (2×10 mL). The aqueous layer was freeze-dried to give 49 mg of4a (95%). The combined organic layers were concentrated, and the residuerefluxed in 3 N HCl for 6 h, then freeze-dried to yield 50 mg of 4c(98%).

[0324] The other two diastereomers, 4b and 4d, were obtained from 3busing the same procedure.

[0325] (2S,3R)-4,4,4-Trifluorovaline (4a)

[0326]¹H NMR (300 MHz, D₂O) δ 4.24 (dd, 1H, J=2.1, 3.9 Hz), 3.23 (m,1H), 1.30 (d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −71.69(d, 3F, J=9.3 Hz); [α]_(D) ^(23.7)=+7.2° (c 0.75, 1 N HCl).

[0327] (2S,3S)-4,4,4-Trifluorovaline (4b)

[0328]¹H NMR (300 MHz, D₂O) δ 4.35 (t, 1H, J=2.7 Hz), 3.27 (m, 1H), 1.22(d, 3H, J=7.5 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −70.04 (d, 3F,J=9.0 Hz); [α]_(D) ²³³+12.8° (c 0.5, 1 N HCl).

[0329] (2R, 3S)-4,4,4-Trifluorovaline (4c)

[0330]¹H NMR (300 MHz, D₂O) δ 4.24 (dd, 1H, J=2.1, 3.9 Hz), 3.23 (m,1H), 1.30 (d, 3H, J=7.2 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −70.04(d, 3F, J=9.0 Hz).

[0331] (2R, 3R)-4,4,4-Trifluorovaline (4d)

[0332]¹H NMR (300 MHz, D₂O) δ 4.35 (t, 1H, J=2.7 Hz), 3.27 (m, 1H), 1.22(d, 3H, J=7.5 Hz); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −71.69 (d, 3F,J=9.3 Hz).

EXAMPLE 16

[0333] N-Boc-5.55-trifluoroleucine Methyl Ester (6)

[0334] A mixture of Boc-DL-trifluoroleucine (1.25 g, 4.38 mmol),iodomethane (0.3 mL, 4.82 mmol), NaHCO₃ (1.1 g, 13.15 mmol), and dry DMF(20 mL) was stirred at room temperature under argon for 6 h, thendiluted with 200 mL of ethyl acetate, and washed with water (4×100 mL).The organic layer was dried over Na₂SO₄ and concentrated to give 1.25 gof product as a pale-yellow oil (95%). Column chromatography on silicagel (500 g) using Et₂O/n-pentane (1:4) as eluant afforded 420 mg of(2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucine methyl ester (6a) (32%),347 mg of (2S,4S)-, (2R,4R)-N-Boc-5,5,5-trifluoroleucine methyl ester(6b) (27%), and 337 mg of the mixture of 6a and 6b (26%).

[0335] (2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucine methyl ester (6a)

[0336]¹H NMR (300 MHz, CDCl₃) δ 5.29 (d, 1H, J=6.9 Hz), 4.32 (m, 1H),3.70 (s, 3H), 2.31 (m, 1H), 2.12 (m, 1H), 1.58 (m, 1H), 1.37 (s, 9H),1.11 (d, 3H, J=6.9 Hz); ¹³CNMR (75.5 MHz, CDCl₃) δ 172.72 (C═O), 155.29(C═O), 128.09 (q, CF₃, ¹J_(CF)=278.9 Hz), 80.27 (C), 52.54 (CH₃), 51.70(CH), 35.13 (q, CH, ²J_(CF)=26.4 Hz), 32.98 (CH₂), 28.30 (3×CH₃), 13.17(CH₃); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −74.15 (d, 3F, J=8.2 Hz);FT-IR (film, v_(max) cm⁻¹) 3360m, 2984m, 2938m, 1747s, 1716s, 1520s,1368s, 1269s, 1168s, 1133m; GC-MS (CI, CH₄): 300 (2, [M+1]+), 284 (7),244 (100), 200 (66), 82 (21), 57 (24).

[0337] (2S,4S)-, (2R,4R)-N-Boc-5,5,5-trifluoroleucine Methyl Ester (6b)

[0338]¹H NMR (300 MHz, CDCl₃) δ 5.02 (d, 1H, J=8.7 Hz), 4.38 (m, 1H),3.76 (s, 3H), 2.32 (m, 1H), 1.91-1.74 (br. m, 2H), 1.44 (s, 9H), 1.20(d, 3H, J=6.9 Hz); ¹³C NMR (75.5 MHz, CDCl₃) δ 173.03 (C═O), 155.86(C═O), 128.24 (q, CF₃, ¹J_(CF)=278.9 Hz), 80.57 (C), 52.80 (CH₃), 50.83(CH), 35.02 (q, CH, ²J_(CF)=26.9 Hz), 33.00 (CH₂), 28.42 (3 ÅCH₃), 12.28(CH₃); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −74.03 (d, 3F, J=8.7 Hz);FT-IR (KBr pellet, v_(max), cm⁻¹) 3368s, 3014m, 2983s, 2961m, 1763s,1686s, 1527s, 1265s, 1214s, 1170s, 1053s, 1028s; GC-MS (CI, CH₄): 300(2, [M+1]⁺), 284 (7), 244 (100), 224 (30), 200 (66), 57 (24).

EXAMPLE 17

[0339] (2S,4R)-, (2R,4S)-N-Ac-5,5,5-trifluoroleucine (7a)

[0340] (2S,4R)-, (2R, 4S)-N-Boc-5,5,5-trifluoroleucinol

[0341] To a solution of 6a (420 mg, 1.4 mmol) in methanol (10 mL) wasadded NaBH4 (531 mg, 14.0 mmol) in small portions. The reaction mixturewas stirred at room temperature for 1 h before removal of the solvent.The residue was partitioned between 100 mL of ethyl acetate and 50 mL ofwater. The aqueous layer was extracted with 100 mL of ethyl acetate. Thecombined organic layers were dried over Na₂SO₄ and concentrated to yield357 mg of the desired product as a white solid (94%). ¹H NMR (300 MHz,CDCl₃) δ 4.74 (m, 1H), 3.71 (m, 2H), 3.58 (m, 1H), 2.31 (m, 1H), 2.14(m, 1H), 1.92 (m, 1H), 1.45 (s, 9H), 1.17 (d, 3H, J=7.0 Hz). ¹³C NMR(75.5 MHz, CDCl₃) δ 156.26 (C═O), 128.41 (q, CF₃, ¹J_(CF)=279.4 Hz),80.14 (C), 64.78 (CH₂), 50.73 (CH), 35.59 (q, CH, ²J_(CF)=29.6 Hz),31.74 (CH₂), 28.52 (3×CH₃), 13.71 (CH₃); ¹⁹F NMR (282.6 MHz,CDCl₃/CFCl₃) δ −73.84 (br. s, 3F); GC-MS (CI, CH₄): 272 (100, [M+1]+),216 (68), 172 (26), 57 (11).

[0342] (2S, 4S)—, (2R, 4R)-N-Boc-5,5,5-trifluoroleucinol

[0343]¹H NMR (300 MHz, CDCl₃) δ 4.58 (m, 1H), 3.79 (m, 1H), 3.68 (m,1H), 3.58 (m, 1H), 2.27 (m, 1H), 2.05 (m, 1H), 1.80 (m, 1H), 1.45 (s,9H), 1.18 (d, 3H, J=6.6 Hz). ¹³C NMR (75.5 MHz, CDCl₃) δ 156.47 (C═O),128.56 (q, CF₃, ¹J_(CF)=278.7 Hz), 80.20 (C), 66.31 (CH₂), 49.49 (CH),35.15 (q, CH, ²J_(CF)=26.7 Hz), 31.71 (CH₂), 28.50 (3×CH₃), 12.56 (CH₃);¹³F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −73.98 (d, 3F, J=8.5 Hz); GC-MS (CI,CH₄): 272 (100, [M+1]⁺), 172 (26), 57 (11).

[0344] (2S,4R)-, (2R,4S)-N-Ac-5,5,5-trifluoroleucine (7a)

[0345] A mixture of (2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucinol (330mg, 1.23 mmol), PDC (4.62 g, 12.3 mmol), and dry DMF (2.5 mL) wasstirred at room temperature under argon for 4 h, then diluted with 50 mLof ethyl acetate and 50 mL of water. The organic layer was washed with30 mL of 1N HCl and 2×30 mL of water, dried over MgSO₄, and concentratedto give 198 mg of (2S,4R)-, (2R,4S)-N-Boc-5,5,5-trifluoroleucine as apale-brownish oil (60%).

[0346] A solution of the above product (180 mg, 0.63 mmol) in 2 mL ofCH₂Cl₂ was treated with 0.5 mL of trifluoroacetic acid for 30 min atroom temperature. After removal of the solvent, the yellowish residuewas dissolved in 2 mL of water, treated with NaOH (126 mg, 3.15) at 0°C., and acetic anhydride (0.12 mL, 1.26 mmol) was then added dropwise.The reaction mixture was stirred at 0° C. for 30 min, then allowed towarm to room temperature. After stirring for another 1 h, the mixturewas diluted with 30 mL of water, acidified to pH 2 with 3 N HCl, andextracted with ethyl acetate (2×90 mL). The combined organic layers weredried over Na₂SO₄ and concentrated to yield 136 mg of 7a as a whitesolid (95%). ¹H NMR (300 MHz, D₂O) δ 4.48 (dd, 1H, J=6.1, 8.8 Hz), 2.51(m, 1H), 2.27 (m, 1H), 2.06 (s, 3H), 1.79 (m, 1H), 1.18 (d, 3H, J=7.0Hz); ¹³C NMR (75.5 MHz, D₂O) δ 175.48 (C═O), 174.60 (C═O), 128.53 (q,CF₃, ¹J_(CF)=278.9 Hz), 51.24 (CH), 34.88 (q, CH, ²J_(CF)=26.6 Hz),31.21 (CH₂), 21.90 (CH₃), 13.03 (CH₃); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H)δ −73.68 (d, 3F, J=9.0 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3343s,3063-2487m (br.), 2932m, 2894m, 1709s, 1613s, 1549s, 1266s, 1179s,1137s; GC-MS (CI, CH₄): 228 (100, [M+1]⁺), 211 (47), 186 (26), 140 (16),57 (11).

[0347] (2S, 4S)- , (2R, 4R)-N-A c-5,5,5-trifluoroleucine (7b)

[0348]¹H NMR (300 MHz, D₂O) 64.48 (dd, 1H, J=3.8, 11.6 Hz), 2.41 (m,1H), 2.07 (s, 3H), 2.15-1.91 (br. m, 2H), 1.16 (d, 3H, J=6.9 Hz); ¹³CNMR (75.5 MHz, D₂O) δ 178.35 (C═O), 177.38 (C═O), 131.09 (q, CF₃,¹J_(CF)=278.3 Hz), 52.72 (CH), 37.31 (q, CH, ²J_(CF)=26.6 Hz), 33.06(CH₂), 24.50 (CH₃), 13.90 (CH₃); ¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ−73.87 (d, 3F, J=8.5 Hz); FT-IR (KBr pellet, v_(max), cm⁻¹) 3336s,2977m, 2949m, 2897m, 2615m, 2473s, 1711 s, 1628s, 1551s, 1276s, 1250s,1127s, 1095s; GC-MS (CI, CH₄): 228 (100, [M+1]⁺), 211 (47), 186 (26),140 (16), 120 (3), 57 (11).

EXAMPLE 18

[0349] (2S,4R)-5.5.5-Trifluoroleucine (8a)

[0350] To a solution of 7a (136 mg, 0.6 mmol) in 2 mL of pH 7.9 aqueousLiOH/HOAc was added porcine kidney acylase I (18 mg) at 27° C. Themixture was stirred at 27° C. for 48 h (pH was maintained at 7.5 byperiodic addition of 1 N LiOH). It was further diluted with 5 mL ofwater, acidified to pH 5.0, heated to 60° C. with Norit, and filtered.The filtrate was acidified to pH 1.5 and extracted with ethyl acetate(2×50 mL). The aqueous layer was freeze-dried to give 63 mg of 8a (95%).The combined organic layers were concentrated, and the residue refluxedin 3 N HCl for 6 h, then freeze-dried to yield 64 mg of 8c (96%).

[0351] The other two diastereomers, 8b and 8d, were obtained from 7busing the same procedure.

[0352] (2S, 4R)-5,5,5-Trifluoroleucine (8a)

[0353]¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −74.33 (d, 3F, J=9.0 Hz);[α]_(D) ^(22.9)=+21.6° (c 0.5, 1N HCl).

[0354] (2S, 4S)-5,5,5-Trifluoroleucine (8b)

[0355]¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) δ −74.11 (d, 3F, J=8.2 Hz);[α]_(D) ²³⁶=−4.0° (c 0.8, 1N HCl).

[0356] (2R,4S)-5,5,5-Trifluoroleucine (8c)

[0357]¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) 5-74.33 (d, 3F, J=9.0 Hz).

[0358] (2R,4R)-5,5,5-Trifluoroleucine (8d)

[0359]¹⁹F NMR (282.6 MHz, D₂O/CF₃CO₂H) 5-74.11 (d, 3F, J=8.2 Hz).

EXAMPLE 19

[0360] Boc-TFV(2S,3 S)-Ser(Ot-Bu)-OMe(2S)

[0361] To a stirred solution of (2S,4S)-5,5,5-Trifluorovaline (4b) (5mg, 0.02 mmol) in DMF (1 mL) was added diisopropylethyl amine (DIEA,0.01 mL, 0.06 mmol), 0-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HBTU, 8 mg, 0.02 mmol), and the HCl salt of(2S)-H-Ser(Ot-Bu)-OMe (9 mg, 0.04 mmol), sequentially. The mixture wasstirred at room temperature for 20 min before dilution with water (5 mL)and extraction with diethyl ether (15 mL). The organic layer was washedwith 1 N HCl (2×5 mL) and 5% NaHCO₃ (2×8 mL), dried over MgSO₄, andconcentrated to give 7 mg of the dipeptide (88%). ¹H NMR (300 MHz,CDCl₃) δ 6.92 (d, 1H, J=7.8 Hz), 5.16 (d, 1H, J=8.7 Hz), 4.65 (m, 1H),4.39 (dd, 1H, J=5.1, 8.8 Hz), 3.81 (dd, 1H, J=2.7, 9.0 Hz), 3.74 (s,3H), 3.56 (dd, 1H, J=3.0, 9.0 Hz), 3.04 (m, 1H), 1.46 (s, 9H), 1.23 (d,3H, J=7.2 Hz), 1.14 (s, 9H); ¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −68.57(d, 3F, J=8.7 Hz).

[0362] Boc-TFV(2S, 3R)-Ser(Ot-Bu)-OMe(2S)

[0363]¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −71.36 (d, 3F, J=7.9 Hz).

[0364] Boc-TFV(2R,3S)-Ser(Ot-Bu)-OMe(2S)

[0365]¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −71.48 (d, 3F, J=8.5 Hz).

[0366] Boc-TFV(2R, 3R)-Ser(Ot-Bu)-OMe(2S)

[0367]¹⁹F NMR (282.6 MHz, CDCl₃/CFCl₃) δ −68.49 (d, 3F, J=9.0 Hz).

[0368] Incorporation by Reference

[0369] All of the patents and publications cited herein are herebyincorporated by reference.

[0370] Equivalents

[0371] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the following claims.

We claim:
 1. A compound represented by A:

wherein X represents O, S, N(R), or C(R)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,or heteroaralkyl; or XR′ represents halide; the stereochemicalconfiguration at any stereocenter of a compound represented by A may beR, S, or a mixture of these configurations; and the enantiomeric excessof a compound represented by A is greater than or equal to about 85%. 2.The compound of claim 1, wherein X represents O or N(R).
 3. The compoundof claim 1, wherein R represents independently for each occurrence H,alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,aralkylaminocarbonyl, or aralkylaminocarbonyl.
 4. The compound of claim1, wherein R represents independently for each occurrence H.
 5. Thecompound of claim 1, wherein R′ represents H, alkyl, or aralkyl.
 6. Thecompound of claim 1, wherein R′ represents H.
 7. The compound of claim1, wherein R represents independently for each occurrence H; and R′represents H.
 8. The compound of claim 1, wherein X represents O orN(R); and R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 9. The compound of claim 1, wherein X represents Oor N(R); and R represents independently for each occurrence H.
 10. Thecompound of claim 1, wherein X represents O or N(R); and R′ representsH, alkyl, or aralkyl.
 11. The compound of claim 1, wherein X representsO or N(R); and R′ represents H.
 12. The compound of claim 1, wherein Xrepresents O or N(R); R represents independently for each occurrence H,alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.
 13. The compound of claim 1, wherein X represents O or N(R);R represents independently for each occurrence H; and R′ represents H,alkyl, or aralkyl.
 14. The compound of claim 1, wherein X represents Oor N(R); R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; and R′ represents H.
 15. The compound of claim 1,wherein X represents O or N(R); R represents independently for eachoccurrence H; and R′ represents H.
 16. A compound represented by B:

wherein X represents O, S, N(R), or C(R)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; or XR′ represents halide;the stereochemical configuration at any stereocenter of a compoundrepresented by B may be R, S, or a mixture of these configurations; andthe enantiomeric excess of a compound represented by B is greater thanor equal to about 85%.
 17. The compound of claim 16, wherein Xrepresents O or N(R).
 18. The compound of claim 16, wherein R representsindependently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl, oraralkylaminocarbonyl.
 19. The compound of claim 16, wherein R representsindependently for each occurrence H.
 20. The compound of claim 16,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.
 21. Thecompound of claim 16, wherein R′ represents H.
 22. The compound of claim16, wherein R represents independently for each occurrence H; and R′represents H.
 23. The compound of claim 16, wherein X represents O orN(R); and R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 24. The compound of claim 16, wherein X representsO or N(R); and R represents independently for each occurrence H.
 25. Thecompound of claim 16, wherein X represents O or N(R); and R′ representsH, aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.
 26. The compound of claim16, wherein X represents O or N(R); and R′ represents H.
 27. Thecompound of claim 16, wherein X represents O or N(R); R representsindependently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 28. The compound of claim 16, wherein X representsO or N(R); R represents independently for each occurrence H; and R′represents H, aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.
 29. The compound of claim16, wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H. 30.The compound of claim 16, wherein X represents O or N(R); R representsindependently for each occurrence H; and R′ represents H.
 31. A compoundrepresented by C:

wherein X represents O, S, N(R), or C(R)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,or heteroaralkyl; or XR′ represents halide; the stereochemicalconfiguration at any stereocenter of a compound represented by C may beR, S, or a mixture of these configurations; and the enantiomeric excessof a compound represented by C is greater than or equal to about 85%.32. The compound of claim 31, wherein X represents O or N(R).
 33. Thecompound of claim 31, wherein R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,aralkylaminocarbonyl, or aralkylaminocarbonyl.
 34. The compound of claim31, wherein R represents independently for each occurrence H.
 35. Thecompound of claim 31, wherein R′ represents H, alkyl, or aralkyl. 36.The compound of claim 31, wherein R′ represents H.
 37. The compound ofclaim 31, wherein R represents independently for each occurrence H; andR′ represents H.
 38. The compound of claim 31, wherein X represents O orN(R); and R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 39. The compound of claim 31, wherein X representsO or N(R); and R represents independently for each occurrence H.
 40. Thecompound of claim 31, wherein X represents O or N(R); and R′ representsH, alkyl, or aralkyl.
 41. The compound of claim 31, wherein X representsO or N(R); and R′ represents H.
 42. The compound of claim 31, wherein Xrepresents O or N(R); R represents independently for each occurrence H,alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.
 43. The compound of claim 31, wherein X represents O orN(R); R represents independently for each occurrence H; and R′represents H, alkyl, or aralkyl.
 44. The compound of claim 31, wherein Xrepresents O or N(R); R represents independently for each occurrence H,alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H. 45.The compound of claim 31, wherein X represents O or N(R); R representsindependently for each occurrence H; and R′ represents H.
 46. A compoundrepresented by D:

wherein X represents O, S, N(R), or C(R)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; or XR′ represents halide;the stereochemical configuration at any stereocenter of a compoundrepresented by D may be R, S, or a mixture of these configurations; andthe enantiomeric excess of a compound represented by D is greater thanor equal to about 85%.
 47. The compound of claim 46, wherein Xrepresents O or N(R).
 48. The compound of claim 46, wherein R representsindependently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl, oraralkylaminocarbonyl.
 49. The compound of claim 46, wherein R representsindependently for each occurrence H.
 50. The compound of claim 46,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.
 51. Thecompound of claim 46, wherein R′ represents H.
 52. The compound of claim46, wherein R represents independently for each occurrence H; and R′represents H.
 53. The compound of claim 46, wherein X represents O orN(R); and R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 54. The compound of claim 46, wherein X representsO or N(R); and R represents independently for each occurrence H.
 55. Thecompound of claim 46, wherein X represents O or N(R); and R′ representsH, aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.
 56. The compound of claim46, wherein X represents O or N(R); and R′ represents H.
 57. Thecompound of claim 46, wherein X represents O or N(R); R representsindependently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 58. The compound of claim 46, wherein X representsO or N(R); R represents independently for each occurrence H; and R′represents H, aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.
 59. The compound of claim46, wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H. 60.The compound of claim 46, wherein X represents O or N(R); R representsindependently for each occurrence H; and R′ represents H.
 61. A compoundrepresented by E:

wherein X represents O, S, N(R), or C(R)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,or heteroaralkyl; or XR′ represents halide; the stereochemicalconfiguration at any stereocenter of a compound represented by E may beR, S, or a mixture of these configurations; and the enantiomeric excessof a compound represented by E is greater than or equal to about 85%.62. The compound of claim 61, wherein X represents O or N(R).
 63. Thecompound of claim 61, wherein R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,aralkylaminocarbonyl, or aralkylaminocarbonyl.
 64. The compound of claim61, wherein R represents independently for each occurrence H.
 65. Thecompound of claim 61, wherein R′ represents H, alkyl, or aralkyl. 66.The compound of claim 61, wherein R′ represents H.
 67. The compound ofclaim 61, wherein R represents independently for each occurrence H; andR′ represents H.
 68. The compound of claim 61, wherein X represents O orN(R); and R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 69. The compound of claim 61, wherein X representsO or N(R); and R represents independently for each occurrence H.
 70. Thecompound of claim 61, wherein X represents O or N(R); and R′ representsH, alkyl, or aralkyl.
 71. The compound of claim 61, wherein X representsO or N(R); and R′ represents H.
 72. The compound of claim 61, wherein Xrepresents O or N(R); R represents independently for each occurrence H,alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H, alkyl,or aralkyl.
 73. The compound of claim 61, wherein X represents O orN(R); R represents independently for each occurrence H; and R′represents H, alkyl, or aralkyl.
 74. The compound of claim 61, wherein Xrepresents O or N(R); R represents independently for each occurrence H,alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H. 75.The compound of claim 61, wherein X represents O or N(R); R representsindependently for each occurrence H; and R′ represents H.
 76. A compoundrepresented by F:

wherein X represents O, S, N(R), or C(R)₂; R represents independentlyfor each occurrence H, alkyl, aryl, heteroaryl, aralkyl, heteroaralkyl,formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; R′ represents H, alkyl, aryl, heteroaryl, aralkyl,heteroaralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; or XR′ represents halide;the stereochemical configuration at any stereocenter of a compoundrepresented by F may be R, S, or a mixture of these configurations; andthe enantiomeric excess of a compound represented by F is greater thanor equal to about 85%.
 77. The compound of claim 76, wherein Xrepresents O or N(R).
 78. The compound of claim 76, wherein R representsindependently for each occurrence H, alkyl, aralkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, aralkylaminocarbonyl, oraralkylaminocarbonyl.
 79. The compound of claim 76, wherein R representsindependently for each occurrence H.
 80. The compound of claim 76,wherein R′ represents H, aralkyl, formyl, acyl, alkoxycarbonyl,aralkoxycarbonyl, alkylaminocarbonyl, or aralkylaminocarbonyl.
 81. Thecompound of claim 76, wherein R′ represents H.
 82. The compound of claim76, wherein R represents independently for each occurrence H; and R′represents H.
 83. The compound of claim 76, wherein X represents O orN(R); and R represents independently for each occurrence H, alkyl,aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 84. The compound of claim 76, wherein X representsO or N(R); and R represents independently for each occurrence H.
 85. Thecompound of claim 76, wherein X represents O or N(R); and R′ representsH, aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.
 86. The compound of claim76, wherein X represents O or N(R); and R′ represents H.
 87. Thecompound of claim 76, wherein X represents O or N(R); R representsindependently for each occurrence H, alkyl, atalkyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl; and R′ represents H, aralkyl, formyl, acyl,alkoxycarbonyl, aralkoxycarbonyl, alkylaminocarbonyl, oraralkylaminocarbonyl.
 88. The compound of claim 76, wherein X representsO or N(R); R represents independently for each occurrence H; and R′represents H, aralkyl, formyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl.
 89. The compound of claim76, wherein X represents O or N(R); R represents independently for eachoccurrence H, alkyl, aralkyl, acyl, alkoxycarbonyl, aralkoxycarbonyl,alkylaminocarbonyl, or aralkylaminocarbonyl; and R′ represents H. 90.The compound of claim 76, wherein X represents O or N(R); R representsindependently for each occurrence H; and R′ represents H.
 91. Thecompound of any of claims 1-90, wherein the enantiomeric excess of saidcompound is greater than or equal to about 90%.
 92. The compound of anyof claims 1-90, wherein the enantiomeric excess of said compound isgreater than or equal to about 95%.
 93. The compound of any of claims1-90, wherein said compound is a single stereoisomer.
 94. The compoundof any of claims 1-93, wherein said compound is in the form of a salt.95. A formulation, comprising a compound of any of claims 1-94; and apharmaceutically acceptable excipient.
 95. An oligopeptide or apolypeptide, comprising a compound of any of claims 1-93.
 96. A methodof resolving into individual enantiomers a mixture of diastereomers of acompound of any of claims 1-90, comprising the steps of: (a) usingchromatography to obtain an individual pair of enantiomers of a compoundof any of claims 1-90 from a mixture of diastereomers of said compound;and (b) using enzymatic hydrolysis to obtain a single enantiomer of saidcompound from the individual pair of enantiomers of said compound. 97.The method of claim 96, wherein (R)₂N represents (alkoxyearbonyl)HN inthe mixture of diastereomers.
 98. The method of claim 96, wherein (R)₂Nrepresents (tert-butyloxycarbonyl)HN in the mixture of diastereomers.99. The method of claim 96, wherein (R)₂N represents (acyl)HN in the theindividual pair of enantiomers subjected to enzymatic hydrolysis. 100.The method of claim 96, wherein (R)₂N represents (acetyl)HN in the theindividual pair of enantiomers subjected to enzymatic hydrolysis. 101.The method of claim 96, wherein the enzyme used is porcine kidneyacylase I.
 102. The method of claim 96, wherein (R)₂N represents(alkoxycarbonyl)HN in the mixture of diastereomers; and (R)₂N represents(acyl)HN in the the individual pair of enantiomers subjected toenzymatic hydrolysis.
 103. The method of claim 96, wherein (R)₂Nrepresents (tert-butyloxycarbonyl)HN in the mixture of diastereomers;and (R)₂N represents (acetyl)HN in the the individual pair ofenantiomers subjected to enzymatic hydrolysis.
 104. The method of claim96, wherein (R)₂N represents (alkoxycarbonyl)HN in the mixture ofdiastereomers; (R)₂N represents (acyl)HN in the the individual pair ofenantiomers subjected to enzymatic hydrolysis; and the enzyme used isporcine kidney acylase I.
 105. The method of claim 96, wherein (R)₂Nrepresents (tert-butyloxycarbonyl)HN in the mixture of diastereomers;(R)₂N represents (acetyl)HN in the the individual pair of enantiomerssubjected to enzymatic hydrolysis; and the enzyme used is porcine kidneyacylase I.
 106. A method of synthesizing a non-native oligopeptide,polypeptide or protein with enhanced hydrophobicity relative to a nativeoligopeptide, polypeptide or protein, comprising the step of using acompound of any of claims 1-93 in place of a leucine or valine in asynthesis of an oligopeptide, polypeptide or protein.
 107. The method ofclaim 106, wherein the synthesis is automated.
 108. A method ofenhancing the hydrophobicity of an oligopeptide, polypeptide or protein,comprising the step of replacing a leucine or valine in an oligopeptide,polypeptide or protein with a compound of any of claims 1-93.
 109. Amethod of synthesizing a trifluoromethyl-containing analogue ofnorvaline or valine, comprising the steps of: (a) oxidizing a protectedserine or homoserine to give an aldehyde; (b) reacting the aldehyde withtrimethyl(trifluoromethyl)silane and fluoride to give a secondaryalcohol; (c) acylating the secondary alcohol using an arylchlorothionoformate to give a thionocarbonate; and (d) reducing thethionocarbonate using a tin hydride and an initiator to give atrifluoromethyl-containing analogue of norvaline or valine.
 110. Amethod of synthesizing a trifluoromethyl-containing analogue of leucine,comprising the steps of: (a) oxidizing a protected homoserine to give analdehyde; (b) reacting the aldehyde withtrimethyl(trifluoromethyl)silane and fluoride to give a secondaryalcohol; (c) oxidizing the secondary alcohol to give a trifluoromethylketone; (d) reacting the trifluoromethyl ketone with(methylene)triphenylphosphine to give an alkene; and (e) hydrogenatingthe alkene to give a trifluoromethyl-containing analogue of leucine.111. In certain embodiments, the present invention relates to a methodof synthesizing protected 5,5,5,5′,5′,5′-hexafluoroleucine, comprisingthe steps of: (a) reacting an oxazolidine aldehyde derived from serinewith a hexafluoroisopropylidene ylide to give an oxazolidine1,1-bis(trifluoromethyl)alkene; (b) hydrogenating the oxazolidine1,1-bis(trifluoromethyl)alkene to give an oxazolidine1,1-bis(trifluoromethyl)alkane; (c) hydrolyzing the oxazolidine1,1-bis(trifluoromethyl)alkane to give a protected amino alcohol; and(d) oxidizing the protected amino alcohol to give protected5,5,5,5′,5′,5′-hexafluoroleucine.
 112. The method of claim 111, whereinthe reagents for step (a) comprise triphenylphosphine and [(CF₃)₂C]₂S₂;the reagents for step (b) comprise hydrogen and 10% palladium on carbon;the reagents for step (c) comprise toluenesulfonic acid and methanol;and the reagents for step (d) comprise pyridinium dichromate.